Methods and compositions related to inhibition of viral entry

ABSTRACT

Disclosed herein are compositions and methods for inhibiting viral entry.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/007,785, filed Feb. 13, 2014; which is a U.S. National StageApplication under 35 U.S.C. 371 of International Application No.PCT/US2012/031015, with an international filing date of Mar. 28, 2012,which claims the benefit of U.S. Provisional Application No. 61/468,094filed Mar. 28, 2011. The aforementioned applications are hereinincorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Number AIR01 076168, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 690181_402D1 SEQUENCE_LISTING.txt. The text fileis 9.3 KB, was created on May 14, 2018, and is being submittedelectronically via EFS-Web.

BACKGROUND

HIV entry is mediated by the viral envelope glycoprotein, whichcomprises non-covalently associated surface (gp120) and transmembrane(gp41) subunits. gp120 is primarily involved in recognition of cellularreceptors, while gp41 directly mediates membrane fusion. When peptidesisolated from the gp41 N- and C-peptide regions (N- and C-peptides) aremixed in solution, they form a six-helix bundle, which represents thepost-fusion gp41 structure. Three N-peptides form a central paralleltrimeric coiled coil (N-trimer) surrounded by three antiparallel helicalC-peptides that nestle into long grooves between neighboring N-peptides.The importance of this structure is indicated by the dominant negativeinhibition of HIV entry by N- and C-peptides.

The available inhibitory and structural data support a working model ofHIV membrane fusion (FIG. 1). Initially, gp120 interacts with cellularCD4 and a chemokine coreceptor (typically CXCR4 or CCR5), causing largeconformational changes in gp120 that propagate to gp41 via thegp41-gp120 interface. gp41 then undergoes a structural rearrangementthat unleashes its N-terminal fusion peptide, which embeds in the targetcell membrane. At this stage of fusion, gp41 adopts an extended“prehairpin intermediate” conformation that bridges both viral andcellular membranes and exposes the N-trimer region. This intermediate isrelatively long-lived (minutes), but ultimately collapses as the N- andC-peptide regions of each gp41 monomer associate to form a hairpinstructure. Three such hairpins (trimer-of-hairpins) form the 6-helixbundle, which forces the viral and cellular membranes into tightapposition and leads to membrane fusion. This structure likelycorresponds to the core of the fusion-active state of gp41 and showssimilarity to the proposed fusogenic structures of envelope fusionproteins from influenza, Moloney Murine Leukemia Virus, and simianimmunodeficiency virus (SIV), and Ebola virus.

According to this model, an inhibitor that binds to the N-trimer andprevents hairpin formation can inhibit viral entry. This has been wellsupported by the discovery of numerous peptide, protein, and smallmolecule inhibitors that bind the N-trimer. A particularly interestingfeature of the N-trimer is the deep hydrophobic “pocket” formed by its17 C-terminal residues. This pocket has several enticing features as aninhibitory target including: (1) a very highly conserved sequence, (2)an essential role in viral entry, (3) a compact binding site vulnerableto inhibition by short peptides, and (4) the availability of severaldesigned peptides (e.g., IQN17, IZN17, 5-helix, N_(CCG)N13 thatauthentically mimic the pocket structure). What is needed in the art arepeptides with suitable pharmacokinetic properties that can inhibit theentry of gp41 into cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, together with the description, illustratecertain embodiments of the disclosed compositions and methods.

FIG. 1 shows an embodiment of a HIV entry pathway. The gp41 fusionpeptide and transmembrane domain are also shown. For clarity, gp120 isomitted from the prehairpin intermediate.

FIG. 2 shows an estimated distance between the N-trimer pocket region(D-peptide binding site) and the target cell membrane.

FIG. 3(a) shows one embodiment of a homotrimeric PEG scaffold asdisclosed herein, and (b) shows one embodiment of a heterotetrameric PEGscaffold as disclosed herein.

FIG. 4(a) shows the observed effects of the PEG linker length onchol-PIE12-trimer potency, and (b) shows the observed effects ofdifferent alkane lengths on C8/C16/C18-PIE12-trimer potency.

FIG. 5 shows the enhanced pharmacokinetic properties of one embodimentof a chol-PIE12-trimer compared with one embodiment of a PIE12-trimer.

DETAILED DESCRIPTION

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a polypeptide is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the polypeptide are discussed, each and every combination andpermutation of polypeptide and the modifications that are possible arespecifically contemplated unless specifically indicated to the contrary.Thus, if a class of molecules A, B, and C are disclosed as well as aclass of molecules D, E, and F and an example of a combination molecule,A-D is disclosed, then even if each is not individually recited, each isindividually and collectively contemplated. Thus, in this example, eachof the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F arespecifically contemplated and should be considered disclosed fromdisclosure of A, B, and C; D, E, and F; and the example combination A-D.Likewise, any subset or combination of these is also specificallycontemplated and disclosed. Thus, for example, the sub-group of A-E,B-F, and C-E are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. This concept applies to all aspects of this applicationincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods, and that each such combination isspecifically contemplated and should be considered disclosed.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. It isunderstood that the disclosed method and compositions are not limited tothe particular methodology, protocols, and reagents described as thesemay vary. It is also to be understood that the terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meanings that would be commonly understood by one of skill inthe art in the context of the present specification.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point “15” aredisclosed, it is understood that greater than, greater than or equal to,less than, less than or equal to, and equal to 10 and 15 are considereddisclosed as well as between 10 and 15. It is also understood that eachunit between two particular units are also disclosed. For example, if 10and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

B. Compositions

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein.

Synthetic C-peptides (peptides corresponding to the C-helix), such asDP178 and C34, are potent inhibitors of HIV-1 membrane fusion and areeffective against both laboratory-adapted strains and primary isolates.Based on the structural features of the gp41 core, these peptides arethought to act through a dominant-negative mechanism, in which exogenousC-peptides bind to the central coiled-coil of gp41 and lead to itsinactivation. These peptides likely act on a pre-hairpin intermediate ofgp41 that forms when the native gp41 structure (i.e., the nonfusogenicconformation present on free virions) is perturbed bygp120/CD4/coreceptor interactions. This pre-hairpin intermediate has anexposed N-coiled-coil, thereby allowing C-peptides to bind andinactivate gp41 prior to the formation of the fusion-active hairpinstructure. Therefore, compounds that bind with high affinity to thiscavity and prevent normal N- and C-helix pairing are effective HIV-1inhibitors. In addition, residues in the cavity are highly conservedamong diverse HIV-1 isolates. Because of the high structuralconservation, drugs targeting this site would have broad activityagainst diverse HIV isolates.

As described herein, the pocket on the surface of the N-helixcoiled-coil of HIV-1 envelope protein gp41 subunit is a drug target.Similarly, cavities on other pathogens (e.g., HIV-2) which can causeAIDS or on pathogens which cause AIDS-like conditions in nonhumanmammals (e.g., SIV) are also drug targets. Available methods (e.g.,mirror image phage display methods, combinational chemistry,computational approaches and other drug screening and medicinalchemistry methods) can be used to identify peptides, D-peptides,including multimers, and peptidomimetics and small molecules that bindthe coiled-coil cavity of HIV-1 (and/or HIV-2) with sufficient affinityto interfere with viral entry into cells and, thus, inhibit viralinfection. Mirror image phage display has been used to identifyD-peptides which bind to a cavity on the surface of the N-helixcoiled-coil of HIV-1 gp41.

Disclosed herein are compositions comprising D-peptides which interactwith the N-trimer pocket of a viral transmembrane protein. For example,the D-peptides can bind to a cavity on the surface of the N-helixcoiled-coil of HIV envelope glycoprotein gp41 (e.g., HIV-1, HIV-2). SuchD-peptides can be of any length, provided that they are of sufficientlength to bind the cavity in such a manner that they interfere with theinteraction of the N-helix coiled-coil cavity and amino acid residues ofthe C-peptide region of viral gp41 and prevent, or inhibit, viral entryinto the cells. For example, the peptide can comprise at least 2, 3, 4,5, 6, 7, 8, 9, or 10 core amino acid residues in length. The amino acidresidues can be naturally occurring or non-naturally occurring ormodified, as described herein. Examples of peptides that bind theN-trimer of HIV gp41 may be found in U.S. application Ser. No.12/526,071, which is incorporated in its entirety by reference herein,and as shown in Table 1.

D-peptides are peptides which are of the opposite handedness from thehandedness of naturally-occurring peptides. Consequently, D-peptides donot serve as efficient substrates for enzymes, and, therefore, are notas readily degraded as L-peptides. In addition, there is no knowneffective immune response which targets D-peptides and therefore, theydo not elicit an immune response comparable to that elicited by L aminoacid peptides. Furthermore, D-peptides have several potential advantagesover L-peptide including: (1) D-peptides are resistant to proteases, aproperty that can dramatically increase serum half-life, (2) L-peptidesmust be injected to avoid digestion, but short D-peptides can beabsorbed systemically when taken orally, and (3) D-peptides represent arich source of structural diversity because they can bind to targetswith unique interface geometries not available to L-peptides.

Examples of D-peptides, identified as described herein, are shown below.In certain embodiments, D-peptides are referred to as Pocket-specificInhibitors of Entry (PIE). An example of such a D-peptide inhibitor isPIE7, which is represented by the sequence Ac-KGACDYPEWQWLCAA-NH2 (SEQID NO: 6). In certain embodiments, one or more N-terminal lysineresidues may be added to a D-peptide to improve water solubility.Particular embodiments of the D-peptides disclosed herein may be shownwith the linker sequence “PEG” before the amino acid sequence.

Disclosed in Table 1 are various examples of D-peptides that can be usedwith the methods and compositions disclosed herein:

TABLE 1 D-peptide binding and neutralization Sample Sequence D10-p5KKGACELLGWEWAWLCAA (SEQ ID NO: 1) 2K-PIE1 KKGACESPEWRWLCAA(SEQ ID NO: 2) 2K-PIE2 KKGACDYPEWRWLCAA (SEQ ID NO: 3) PIE2-AAAKGACDYPEWRWLCAAA (SEQ ID NO: 4) PIE2 KGACDYPEWRWLCAA (SEQ ID NO: 5) PIE7KGACDYPEWQWLCAA (SEQ ID NO: 6) PIE8 KGACDYKEWQWLCAA (SEQ ID NO: 7)PEG-PIE7 PEG-KGACDYPEWQWLCAA (SEQ ID NO: 8) PEG-(PIE7)₂PEG-(KGACDYPEWQWLCAA)2 (SEQ ID NO: 9) 2K-PhD1 KKGACPREWHWLCAA(SEQ ID NO: 10) PhD1 GACPREWHWLCAA (SEQ ID NO: 11) 2K-PIE0KKGACDYWEWRWLCAA (SEQ ID NO: 12) D-PIE2 DGACDYPEWRWLCAA (SEQ ID NO: 13)2K-PIE3 KKGACDDPDWQWLCAA (SEQ ID NO: 14) 2K-PIE4 KKGACEDPDWQWLCAA(SEQ ID NO: 15) 2K-PIE5 KKGACEDPEWQWLCAA (SEQ ID NO: 16) 2K-PIE6KKGACNDPEWQWLCAA (SEQ ID NO: 17) PIE1 DGACESPEWQWLCAAGAA (SEQ ID NO: 18)R4#9 ACPPEWHWLCGGGSA (SEQ ID NO: 19) R4#12 ACPVEWRWLCGGGSA(SEQ ID NO: 20) R4#6 ACPIEWRWLCGGGSA (SEQ ID NO: 21) PhD1bACPREWHWLCGGGSA (SEQ ID NO: 22) PIE7-GK GACDYPEWQWLCAAGK (SEQ ID NO: 23)PIE7-GKK GACDYPEWQWLCAAGKK (SEQ ID NO: 24) K-PIE7-GK KGACDYPEWQWLCAAGK(SEQ ID NO: 25) PIE12 HPCDYPEWQWLCELGK (SEQ ID NO: 26) PIE13HPCDYPEWQWLCKLGK (SEQ ID NO: 27) PIE14 HPCDYPEWQWLCRLGK (SEQ ID NO: 28)PIE15 HACDYPEWQWLCELGK (SEQ ID NO: 29) Consensus sequence CDYXEWXWLC(SEQ ID NO: 33) Consensus sequence CX₅EWXWLC (SEQ ID NO: 34)Consensus sequence CX₃EWXWLC (SEQ ID NO: 35) Consensus sequence CX₄WXWLC(SEQ ID NO: 36)

The term “D-amino acid residue”, as used herein, refers to an α-aminoacid residue having the same absolute configuration as D-glyceraldehyde.

Embodiments of the compositions disclosed herein comprise peptides,portions of the peptides, and variations/derivatives of the peptidesthat can be used as inhibitors of HIV entry into cells. Particularembodiments of the peptides disclosed herein, or a portion of suchpeptides, that is sufficient to fit into the hydrophobic pocket at theC-terminal end of the coiled-coil and prevent interaction of theC-peptide region with the N-peptide region of gp41, may be useful toinhibit HIV infection. A portion of any of the peptides represented orof a derivative thereof can be from 2 to 20 (any number of residues from2 to 20) amino acid residues in size. In specific embodiments,D-peptides which comprise at least the consensus sequence EWXWL (SEQ IDNO: 30) or at least the sequence WXWL (SEQ ID NO: 31), can be used.Where D-peptides as described herein include amino acid residues inaddition to a consensus sequence, the additional amino acid residues andthe size of the D-peptides can be selected with reference to thepeptides described herein or can be designed independent of thosepeptides, provided that peptide can fit into the hydrophobic pocket andact as an inhibitor. Additional amino acid residues can also be presentat the N-terminus, the C-terminus or both of the D-peptides describedherein, thus producing a larger peptide. Alternatively, there can beother amino acid residues selected, for example, to enhance bindingaffinity. For example, such a peptide can include the conserved aminoacid residues, which can be at the same positions as those at which theyoccur in the peptides disclosed herein. In some embodiments, the peptidecan comprise the core sequence “WXWL” (SEQ ID NO: 31).

In some embodiments of the peptides disclosed herein, the peptides maycomprise amino acid residues which can be different from the amino acidresidues at these positions in any of the peptides disclosed herein(e.g., can be isoleucine or asparagine or other amino acid residue whichdoes not appear in the peptides disclosed herein) or can be substitutedfor or replaced by an amino acid residue represented at a specificposition in another peptide. Amino acid residues other than theD-versions of the 20 L-amino acids found in natural proteins can beused. Such changes can be made, for example, to enhance bioavailability,binding affinity or other characteristic of the peptide. A D-peptide cancomprise the conserved amino acid residues present in the peptidesdisclosed herein, but they can be separated by fewer (or more) aminoacid residues than the number of intervening amino acid residues shownin Table 1. For example, fewer than five amino acid residues can bepresent between the first cysteine and the glutamic acid in theconsensus sequence. Alternatively, these two residues can be separatedby more than five amino acid residues. Internal modifications can alsobe made (e.g., to enhance binding or increase solubility of a peptide).For example, the first leucine of D10p5 can be replaced by an arginineto increase solubility. A D-peptide can have additional moieties oramino acids at its N-terminus. For example, a moiety which blocks the Nterminus or gets rid of the charge otherwise present at the N-terminuscan be added. The moiety can be, for example, a blocking moiety, such asan acetyl group linked directly to the glycine (G), or an acetyl grouplinked to one or more additional amino acid residues linked to theN-terminal of G, such as an acetyl group linked to one or more lysineresidues, which, in turn, are linked to the N-terminal G.

In one embodiment of the peptides disclosed herein, two lysine residuesare linked to the N-terminal G (KKGAC . . . , SEQ ID NO: 32), forexample to increase the solubility of the peptide, and then a blockingmoiety, such as an acetyl group, can be linked to the terminal lysine(acetyl group-KKGAC . . . , SEQ ID NO: 32). In another embodiment, fourlysine residues are linked to the N-terminal G. In addition, a D-peptidecan have additional and/or altered moieties or amino acids at itsC-terminus. For example, one or both of the alanine residues at theC-terminus can be altered and/or one or more residues can be added atthe C-terminus, for example to enhance binding. Alternatively,functional (chemical) groups other than amino acid residues can beincluded to produce an inhibitor of the embodiments disclosed herein.For example, these additional chemical groups can be present at theN-terminus, the C-terminus, both termini or internally.

Two or more D-peptides can be linked via an appropriate linker (e.g., alinker of amino acid residues or other chemical moieties) to increasethe effectiveness of inhibition. Alternatively, one or more D-peptidescan be linked via an appropriate linker to a molecule (drug) that bindsto HIV gp120, CD4, CCR5, CXCR4, or a non-pocket region of HIV gp41 toincrease the effectiveness of inhibition.

Regarding the nomenclature of the peptides disclosed herein, differentfamilies of peptides are referred to as x-mers, where x is consideredthe number of residues between the cysteine residues. The x-mers arereferred to as the “core peptides.” For example, SEQ ID NO: 6(KGACDYPEWQWLCAA) is comprised of 15 residues, and so in the standardart would be referred to as a 15-mer. However, in certain embodimentsdisclosed herein, the length of residues between the cysteines (C) is 8,so it would be considered an 8-mer (and referred to as having 8 coreresidues), and referred to as such throughout the application. Inparticular embodiments, amino acids outside of the two Cys residues arereferred to as “flanking” sequences. This naming scheme allows differentfamilies of peptides that differ in the number of residues between thetwo Cys residues, but can vary in total peptide length due todifferences in their flanking sequences, to be distinguished. Forexample, SEQ ID NO: 6 (KGACDYPEWQWLCAA) has a length of 15 residues, isa member of the 8-mer peptide family (as it has 8 core residues), andhas an N-terminal flanking sequence of KGA and a C-terminal flankingsequence of AA. In comparison, SEQ ID NO: 2 (KKGACESPEWRWLCAA) has atotal peptide length of 16 residues, but is also a member of the 8-merpeptide family and contains an N-terminal flanking sequence of KKGA anda C-terminal flanking sequence of AA. In addition to the core residuesand flanking residues present on the peptides disclosed herein, all ofthe peptides disclosed herein may comprise blocked N- and C-termini withthe N-termini being blocked by an acetyl group (Ac) and the C-terminibeing blocked by an amino group (NH₂).

As described herein, the D-peptides of the present disclosure can beflanked by GA at the N-terminus and AA at the C-terminus, due to thedesign of the library used in identifying the D-peptides. Some or all ofthese amino acid residues may be altered, replaced or deleted in orderto produce D-peptides with, for example, altered absorption,distribution, metabolism and/or excretion. In one embodiment, theC-terminus is modified by the addition of a glycine residue immediatelybefore the C-terminal amide. In another embodiment, the most C-terminalA is altered/modified or replaced by a different amino acid residue ordeleted. In yet a further embodiment, amino acids are added to theC-terminus and/or N-terminus. Thus, it is contemplated herein that theboth the N-terminal GA and C-terminal AA can substituted or additionallyflanked to enhance potency. For example one or two lysines can be addedto the C-terminal AA to create single or double lysine variants of aparticular PIE. Also for example, the N-terminal Lys can be modified tocomprise HP at the N-terminus.

One sequence of a D-peptide contemplated by the present disclosure isAc-HPCDYPEWQWLCELGK-NH2 (SEQ ID NO: 26) which is also referred to asPIE12. In another embodiment, the D-peptide may be PIE7-GK with asequence of Ac-GACDYPEWQWLCAAGK-NH2 (SEQ ID NO: 23). This peptide is thesame as PIE7, except that the Lys has been moved to the C-terminus. Themove results in slightly enhanced potency and allows for thecrosslinking of peptides via their C-termini. Another example of a PIE7variant includes PIE7-GKK (GACDYPEWQWLCAAGKK, SEQ ID NO: 24). This is adouble Lys variant of PIE7-GK, and may serve as a central peptide incertain embodiments of a trimeric PIE7 (the central PIE7-GKK isconnected to two flanking PIE7-GK peptides). These connections are allvia the C-terminus. Also disclosed is K-PIE7-GK (KGACDYPEWQWLCAAGK, SEQID NO: 25). This double Lys variant of PIE7-GK can serve as a centralpeptide in particular embodiments of other embodiments of trimeric PIE7(the central K-PIE7-GK is connected to two flanking peptides—PIE7-GK andPIE7). These connections link the N- to C-termini of neighboringpeptides. Additional examples of variant peptides disclosed herein arethe following variants of PIE12: PIE13, HPCDYPEWQWLCKLGK (SEQ ID NO:27); PIE14, HPCDYPEWQWLCRLGK (SEQ ID NO: 28); and PIE15,HACDYPEWQWLCELGK (SEQ ID NO: 29).

In certain embodiments, the peptides disclosed herein can also bepresent as multimers, such as dimers or trimers. For example, when themultimer is a dimer, the dimer can be comprised of two identicalpeptides, or can be comprised of two different peptides. Alternatively,the multimer can also be a trimer. When the multimer is a trimer, thetrimer can be comprised of two identical peptides and one differentpeptide, or three identical peptides, or three different peptides, eachof which is distinct from each other.

1. Multimers

Disclosed herein are multimers of the peptides which are describedherein. In certain embodiments, the multimers disclosed herein cancomprise at least one D-peptide which interacts with the N-trimer pocketof a viral transmembrane protein. The multimer can be a dimer, trimer,or higher order multiples such as a tetramer, but could also includemultimers with 5, 6, 7, 8, 9, 10, 11, or 12 D-peptides. Thus disclosedherein are compositions comprising multimers including one or moreD-peptide pocket-specific inhibitors of entry (PIE).

It is understood and herein contemplated that the disclosed D-peptidescan be crosslinked to form multimers. In certain embodiments, themultimers may be crosslinked through the use of multimer scaffolds. Anexample of a crosslinker is polyethylene glycol (PEG) derivatized withNHS-ester (reacts with Lys) or maleimide (reacts with Cys). In otherembodiments, crosslinkers can also contain two distinct linkagechemistries (e.g., NHS-ester on one end and maleimide on the other end).In particular embodiments, D-peptides may also be linked by directdisulfide bond formation between two Cys residues.

In certain embodiments, the multimer scaffold can be a trimeric scaffoldcomprising three NHS ester groups. In particular embodiments, themultimer scaffold may be a homotrimeric scaffold or a heterotrimericscaffold comprising three NHS ester groups. Furthermore, in otherembodiments, the multimer scaffold may be a tetrameric scaffoldcomprising three NHS ester groups and a fourth orthogonal group. In suchembodiments, the multimer scaffold may be a heterotetrameric scaffoldcomprising three NHS ester groups and a fourth orthogonal group.Additionally, particular embodiments of the disclosed crosslinker andmultimer scaffold can comprise a tris, di-lysine, benzene ring,phosphate, or peptide core. Other crosslinkers disclosed herein for usewith the disclosed compositions comprise thiol-reactive groups, e.g.,haloacetyls (e.g., iodoacetate), pyridyl disulfides (e.g., HPDP), andother thiols.

The D-peptides that are linked can be any of those disclosed herein, andthe D-peptides can be identical to each other or can each be different.When a dimer is present, the N-termini of both of the D-peptides can becrosslinked to each other. Alternatively, the C-termini of theD-peptides can be crosslinked. Also, the N-terminus of one D-peptide andthe C-terminus of the other D-peptide are crosslinked. When a trimer ispresent, the N-termini and C-termini of the D-peptides can be linked inany combination. For example, they can be linked in any of the followingarrangements: N—N/C—C-peptide 1's N-terminus links to peptide 2'sN-terminus; peptide 2's C-terminus links to peptide 3's C-terminus.Using this naming, there are 16 possible trimer lineages: X/Y where Xand Y═N—N, N—C, C—N, or C—C. D-peptides can also be linked to a centralscaffold by the N- or C-termini or an internal location or a combinationof these. Thus, for example, it is contemplated herein that one or moreD-peptides can be crosslinked at internal residues rather than aterminal crosslinking. It is further contemplated that in trimers aninternal crosslinker can be used for one peptide pair (e.g., peptide 1to peptide 2) and a terminal crosslinker (N- or C-termini) can be usedfor crosslinking peptide 2 to peptide 3.

As used herein, the naming scheme for multimers describes the way thepeptides are connected. For example, C5C-PIE7-trimer means that threePIE7 peptides are connected via C- to C-terminal connections using aPEG₅ spacer. N9C-PIE7-trimer means that three PIE7 peptides areconnected via N- to C-terminal connections using a PEG₉ spacer. Someexamples of dimers are as follows: N9C-PIE7-dimer, C9C-PIE7-dimer,N5N-PIE7-dimer, N5C-PIE7-dimer, C5C-PIE7-dimer, N0N-PIE7-dimer,N0C-PIE7-dimer, and C0C-PIE7-dimer. Note: The zero length spacers can beany of a variety of short crosslinkers (e.g., BS3, DSG, or DST). Thestructure of DSG is as follows:

In some embodiments of the compositions disclosed herein, the C5Cconnection geometry can be used as the linkage for making dimers andtrimers. Examples of such dimers include C5C-PIE12-dimer andPEG₅-PIE13-dimer (this peptide has an internal Lys residue, andtherefore a dimer can be made by crosslinking via this internal Lys). Incertain embodiments, a PEG₅ linker can be used, for example. Examples oftrimers include: C5C-PIE7-trimer, C5C-PIE12-trimer, and theC0C-PIE7-trimer.

As used herein, the term “PIE12-trimer” is a generic term for a multimerthat represents a number of molecules with slightly different chemicalcompositions in which three PIE12 monomers are linked together byvarious crosslinking strategies. In certain embodiments, one class ofPIE12-trimer may be constructed by connecting monomers using PEGcrosslinkers of various lengths without use of a central scaffold. Insuch embodiments, the trimers may be designated, for example,CxC-PIE12-trimer where “CxC” represents linkage of PIE12 monomers via aunique primary amine of a lysine side chain where the lysine residue islocated at the C-terminus of the peptide monomer. In other embodiments,NxN-PIE12-trimers represent linkage by a lysine located at theN-terminus. The “x” in this context refers to the number of PEG units inthe crosslinker connecting individual monomers. In particularembodiments, a central monomer containing two lysines may be used tomake trimers of this type. An alternate name for trimers of this typeis, for example, C5C(PIE12)₃ where the “3” subscript indicates a trimer.

As described herein, some embodiments of PIE12-trimers may beconstructed using a central multimer scaffold containing a trivalentatom (i.e., nitrogen) at its core with three PEG linkers or “arms” ofvarious length connecting PIE12 monomers into a trimer. In otherembodiments, the central multimer scaffold may comprise the use of atetravalent atom at the core of the multimer scaffold (i.e., carbon),with, for example, three PEG linkers of various lengths connectingindividual monomers.

In certain embodiments, potency-enhancing versions of PIE12-trimer maybe assembled using a carbon core scaffold in which a potency-enhancingcargo moiety is attached to a PIE12-trimer utilizing the fourth arm ofthe tetravalent scaffold. In such embodiments, PEG units of variouslength (i.e, 2-132 PEG units) can be used to link various moieties tothe 4th arm. One example of a PIE12-trimer is chol-PEG₂₄-PIE12-trimer,where “chol” is short for thiocholesterol and “PEG₂₄” refers to thenumber of PEG units comprising the 4th arm. In particular embodiments,the potency-enhancing cargo can be attached to the 4th arm PEG unit byvarious chemical reactivities including maleimide chemistry. Thisnomenclature for trimerization applies to other D-peptides describedherein (e.g., PIE7 or PIE7-GK).

The multimers disclosed herein can be made of any combination ofpeptides, including those disclosed in Table 1, or variants thereof,such that the multimers can inhibit viral entry into a cell. In certainembodiments, the multimers can be made up of one of the peptidesdisclosed herein, two of the peptides disclosed herein, or three or moreof the peptides disclosed herein. In such embodiments, all of thepeptides can be identical, or they can be any combination of peptides,including those disclosed and those which are not specificallydisclosed. In particular embodiments, at least one of the peptides cancomprise the sequence WXWL (SEQ ID NO: 31). In other embodiments, themultimers disclosed herein can be made up of at least one D-peptide, twoof more different D-peptides, or other components as well.

a) Multimer Scaffold

As an alternate strategy for making multimers, a central multimericscaffold can be used to attach one or more D-peptides. In particularembodiments, a multimeric scaffold as disclosed herein may comprise acentral trifunctional crosslinker tris(succinimidyl) aminotriacetate,such as TSAT, which contains three N-hydroxysuccinimide (NHS) estergroups. In some embodiments, this geometry is referred to as “the claw”,as the configuration resembles an eagle claw. Two examples of thisstrategy are (1) a short claw (which directly links TSAT to thepeptides) and (2) a long claw (which uses an extended form of TSAT(LC-TSAT) that contains an additional six-atom spacer between TSAT andthe peptides). Other spacer lengths or compositions (e.g., PEG) can alsobe used. Examples different claw configurations include PIE7-GK (longclaw) and PIE7-GK (short claw).

Below is a representation of LC-TSAT:

And the following is a representation of TSAT:

“Over-engineering” future D-peptides means improving affinity even afterreaching the potency limit. Such inhibitors do not show improvedanti-viral potency in vitro, but have a reserve of binding energy(affinity) that acts as a “resistance capacitor” to defend againstpotential resistance mutations (i.e., resistance mutations thatmoderately affect binding affinity would have no effect on potency).This “resistance capacitor” property discourages the stepwiseaccumulation of multiple subtle mutations that combine to conferresistance. Individual mutations have no effect on inhibitor potency anddo not confer a growth advantage in the presence of inhibitors. This“resistance capacitor” may be especially beneficial for trimericD-peptide inhibitors, because resistance mutations simultaneously affectall three pockets. In certain embodiments, as a further defense againstthe development of resistance, the trimeric D-peptides disclosed hereincan also be constructed by using three different D-peptide sequences,each with a distinct resistance profile. Such a heterotrimer wouldpresent a significant additional barrier to the development ofresistance.

b) Heterotetramer

As disclosed herein, the PIE12-trimer is a potent inhibitor of HIVentry. In certain embodiments of the compositions disclosed herein,further modifications are contemplated that allow for PIE12 to, 1) besynthesized more easily and in higher yield; 2) to possess enhancedpharmacokinetic properties (e.g., by reducing renal filtration since itis smaller than the glomerular filtration cutoff molecular weight); and3) to allow for local concentration on the cell surfaces where HIV entrytakes place, improving potency by overcoming the kinetic potency limit.In particular embodiments, to produce PIE12-trimer variants with some orall of these improved properties, a custom-designed heterotetrameric PEGscaffold can be employed. This scaffold typically has three arms withone type of reactive group (e.g., NHS ester) for attachment of the PIED-peptide. A fourth group, typically with a longer PEG arm, has areactive group orthogonal to the other three arms (e.g., maleimide ifthe three arms have NHS esters). This modular heterotetramer scaffolddesign allows straightforward modification of any of the PEG arm lengthsand significantly simplifies synthesis of trimeric PIEs with appendedpotency-enhancing cargoes. Below is an example of a heterotetrameric PEGscaffold according to the present description.

In certain embodiments, the disclosed compositions comprise a multimerscaffold, such as a heterotetramer scaffold, that can be modified tocomprise a potency-enhancing cargo. As used herein, a potency-enhancingcargo is a cargo enhances the potency of the compositions disclosedherein. In some embodiments, a potency-enhancing cargo comprises a cargothat has pharmacokinetic-enhancing properties. In other embodiments, apotency-enhancing cargo comprises a cargo that has membrane-localizingproperties. In particular embodiments, the potency-enhancing cargo maycomprise a pharmacokinetic-enhancing cargo including any group that willreduce clearance of the peptide. For example, disclosed herein arecompositions comprising a multimer scaffold with a potency-enhancingcargo, wherein the potency-enhancing cargo is a sterol (e.g.,cholesterol), albumin, polyethylene glycol, a sugar, maltose bindingprotein, serum albumin, ubiquitin, streptavidin, immunoglobulin domains,keyhole limpet hemacyanin, sperm whale myoovalbumin, bovine pancreatictrypsin inhibitor, green fluorescent protein, gold particles, magneticparticles, agarose beads, lactose beads, or fatty acids. In otherembodiments, the potency-enhancing cargo can be the linking of multiplemultimers such as the linking of multiple trimers (to increase molecularweight and reduce renal filtration). Thus, for example, disclosed hereinare compositions comprising one or more D-peptide pocket-specificinhibitors of entry (PIE), a multimer scaffold, and a potency-enhancingcargo, wherein the potency-enhancing cargo is cholesterol or an analogthereof.

In particular embodiments, the compositions disclosed herein include aPIE12-timer with a pharmacokinetic-enhancing cargo exemplified as shown:

Also disclosed herein are PEG linkers. In certain embodiments, thePEGylation that generates a multimer can result in a PEG linker ofvarying lengths. In particular embodiments, the use of such PEG linkersprovides space between the potency-enhancing cargo and the D-peptidepocket-specific inhibitors of entry. It is understood and hereincontemplated that the length of the PEG linker can improve IC₅₀ and thehalf-life of the composition. However, too bulky a linker can also havedetrimental effects. Thus, disclosed herein are compositions wherein thePEG linker is a linker between the potency-enhancing cargo and D-peptidepocket-specific inhibitors of entry comprising between 12 and 48ethylene glycol repeats. Accordingly disclosed herein are PEG linkerswith 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48 ethylene glycol repeats, referred to as PEG₁₂, PEG₁₃, PEG₁₄,PEG₁₅, PEG₁₆, PEG₁₇, PEG₁₈, PEG₁₉, PEG₂₀, PEG₂₁, PEG₂₂, PEG₂₃, PEG₂₄,PEG₂₅, PEG₂₆, PEG₂₇, PEG₂₈, PEG₂₉, PEG₃₀, PEG₃₁, PEG₃₂, PEG₃₃, PEG₃₄,PEG₃₅, PEG₃₆, PEG₃₇, PEG₃₈, PEG₃₉, PEG₄₀, PEG₄₁, PEG₄₂, PEG₄₃, PEG₄₄,PEG₄₅, PEG₄₆, PEG₄₇, PEG₄₈, respectively.

It is also understood that PEG linkers can be used to link the PIE armsto the scaffold. In certain embodiments, in linking the D-peptidepocket-specific inhibitors of entry to the scaffold, the PEG linkers cancomprise 2, 3, 4, 5, 6, 7, or 8 ethylene glycol repeats.

Thus, it is understood that the disclosed compositions can comprise theculmination of all the features disclosed herein such as one or moreD-peptides, multimer scaffolding, potency-enhancing cargo, andmodification of the flanking regions of D-peptides, and PEG linkers.Accordingly, disclosed herein are compositions comprising one or moreD-peptides and a potency-enhancing cargo, wherein the one or moreD-peptides are linked by a multimer scaffold, wherein the multimerscaffold is linked to the D-peptides via a PEG linker, and wherein thepotency-enhancing cargo is linked to the multimer scaffold via a PEGlinker.

The multimer scaffold as disclosed herein may be use for a multimerscaffold-based design method for multimeric D-peptide drug optimization(both peptide geometry and localization to the site of action viaconjugated localizing cargoes). In certain embodiments, multimerscaffold-based design allows for alterations in the scaffold toaccommodate a variety of cargoes and chemistries (e.g., “click”chemistry), as well as rapid optimization of PEG arm lengths. Forexample, for viruses that undergo membrane fusion within the endosome,such as HIV and Ebola, the multimer scaffold-based strategies disclosedherein could be employed to identify and attach an endosome-targetingmoiety to localize an inhibitor to the site of virus entry and increaseinhibitor potency. Additionally, particular embodiments of the multimerscaffold-based strategy as disclosed herein may allow for theidentification of, and conjugation to a variety of potency-enhancingcargoes to modulate pharmacokinetic properties (e.g., large branchedPEGs, albumin, or albumin-binding peptides) and membrane localization.

c) Avidity of Multimers

Disclosed herein are compositions comprising a multimer as disclosedherein and an N-trimer molecule, wherein the multimer, when associatedwith the N-trimer molecule, has an increased affinity for the N-trimermolecule, when compared with the affinity of a single peptide, orcontrol peptide, for the N-trimer molecule. The single peptide, orcontrol peptide, can be identical to one of the components of themultimer, or the single peptide can be a different peptide which is notcontained in the multimer.

The multimer can exhibit about a 2-fold, 3-fold, 4-fold, 5-fold,10-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold,200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, 2000-fold, 3000-fold,4000-fold, 5000-fold, or 10,000-fold increase in affinity for theN-trimer when compared with the affinity of one of the components of themultimer alone.

The multimer can have any of the characteristics or properties that aredisclosed herein. Any of the multimers disclosed herein are capable ofhaving avidity as described herein, and any of them can be used with themethods disclosed herein for increasing inhibition of viral entry.

d) Peptide Variants

Also disclosed herein are variants of the peptides described herein andthat are herein contemplated. Peptide variants and derivatives thereofare well understood to those of skill in the art and in can involveamino acid sequence modifications. Those peptides disclosed herein thatcan be used to inhibit viral entry can comprise such amino acid sequencemodifications. One of skill in the art would be able to readilydetermine which modifications can be made in order to retain theactivity of the peptide.

Analogs of the peptides disclosed herein are also contemplated. Theseanalogs include one or more D-amino acids of the peptide structure whichare substituted with a homologous amino acid such that the properties ofthe original peptide are maintained. In certain embodiments,conservative amino acid substitutions may be made at one or more aminoacid residues. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art, including basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Non-limiting examples of homologoussubstitutions that can be made in the peptide structures of the peptidesdisclosed herein include substitution of D-phenylalanine withD-tyrosine, D-pyridylalanine or D-homophenylalanine, substitution ofD-leucine with D-valine or other natural or non-natural amino acidhaving an aliphatic side chain and/or substitution of D-valine withD-leucine or other natural or non-natural amino acid having an aliphaticside chain. This is given as an example and is not intended to belimiting. One of skill in the art would be capable of makingconservative substitutions to a D-peptide.

It is further understood that each D-peptide disclosed herein comprisesparticular residues that contact the pocket of the deep groove in theN-trimer region of gp41. For example, residues 2, 3, 4, 8, 9, 11, 12,and 15 are contact residues of PIE7 and residues 2, 3, 7, 8, 10, 11, and14 are contact residues of PIE12. In both PIE7 and PIE12 the residuescorresponding to E, W, W and L form the core sequence EWXWL (SEQ ID NO:30) and comprise the internal most contact residues (residues 8, 9, 11,and 12 for PIE7 and residues 7, 8, 10, and 11 for PIE12). It iscontemplated herein that making substitutions at the contact residuescan significantly affect the binding affinity of the D-peptide for thedeep groove. Residues more amenable to change are non-contact residuesand contact residues at the C-terminal and N-terminal ends of thepeptide.

It is understood that the description of conservative mutations andhomology can be combined together in any combination, such asembodiments that have at least 70% homology to a particular sequencewherein the variants are conservative mutations.

The opposite stereo-isomers of naturally occurring peptides aredisclosed, as well as the stereo-isomers of peptide analogs. These aminoacids can readily be incorporated into polypeptide chains by chargingtRNA molecules with the amino acid of choice and engineering geneticconstructs that utilize, for example, amber codons, to insert the analogamino acid into a peptide chain in a site specific way (Thorson et al.,Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion inBiotechnology, 3:348-354 (1992); Ibba, Biotechnology & GeneticEngineering Reviews 13:197-216 (1995), Cahill et al., TIBS,14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba andHennecke, Bio/technology, 12:678-682 (1994) all of which are hereinincorporated by reference at least for material related to amino acidanalogs).

Molecules can be produced that resemble peptides, but which are notconnected via a natural peptide linkage. For example, linkages for aminoacids or amino acid analogs can include CH2NH—, —CH2S—, —CH2-CH2-,—CH═CH— (cis and trans), —COCH2-, —CH(OH)CH2-, and —CHH2SO— (these andothers can be found in Spatola, A. F. in Chemistry and Biochemistry ofAmino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker,New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1,Issue 3, Peptide Backbone Modifications (general review); Morley, TrendsPharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res14:177-185 (1979) (—CH2NH—, CH2CH2-); Spatola et al. Life Sci38:1243-1249 (1986) (—CH H2-S); Hann J. Chem. Soc Perkin Trans. I307-314 (1982) (—CH—CH—, cis and trans); Almquist et al. J. Med. Chem.23:1392-1398 (1980) (—COCH2-); Jennings-White et al. Tetrahedron Lett23:2533 (1982) (—COCH2-); Szelke et al. European Appln, EP 45665 CA(1982): 97:39405 (1982) (—CH(OH)CH2-); Holladay et al. Tetrahedron. Lett24:4401-4404 (1983) (—C(OH)CH2-); and Hruby Life Sci 31:189-199 (1982)(—CH2-S—); each of which is incorporated herein by reference. Analternative non-peptide linkage is —CH2NH—. It is understood thatpeptide analogs can have more than one atom between the bond atoms, suchas b-alanine, g-aminobutyric acid, and the like.

Amino acid analogs and peptide analogs often have enhanced or desirableproperties, such as, more economical production, greater chemicalstability, enhanced pharmacological properties (half-life, absorption,potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum ofbiological activities), reduced antigenicity, and others.

2. Pharmaceutical Carriers/Delivery of Pharmaceutical Products

The peptides and multimers disclosed herein (alternatively referred toas compositions) can also be administered in vivo in a pharmaceuticallyacceptable carrier. By “pharmaceutically acceptable” is meant a materialthat is not biologically or otherwise undesirable, i.e., the materialmay be administered to a subject, along with the peptide disclosedherein, without causing any undesirable biological effects orinteracting in a deleterious manner with any of the other components ofthe pharmaceutical composition in which it is contained. The carrierwould naturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art.

The compositions may be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, by subcutaneous injection, transdermally, extracorporeally,topically or the like, including topical intranasal administration oradministration by inhalant. As used herein, “topical intranasaladministration” means delivery of the compositions into the nose andnasal passages through one or both of the nares and can comprisedelivery by a spraying mechanism or droplet mechanism, or throughaerosolization. Administration of the compositions by inhalant can bethrough the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of thecompositions required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the disease, its mode of administration and the like. Thus,it is not possible to specify an exact amount for every composition.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem (i.e., depot) such that a constant dosage is maintained. See,e.g., U.S. Pat. No. 3,610,795, which is incorporated by referenceherein.

a) Pharmaceutically Acceptable Carriers

The compositions, including peptides and multimers thereof, can be usedtherapeutically in combination with a pharmaceutically acceptablecarrier.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionmay be from about 5 to about 8, and alternatively from about 7 to about7.5. Further carriers include sustained release preparations such assemipermeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the route of administration andconcentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,anti-inflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration may be topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection. The disclosedpeptides and multimers thereof can be administered intravenously,intraperitoneally, intramuscularly, subcutaneously, intracavity, ortransdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable. Additionally, it iscontemplated herein that compositions designed for oral administrationcan further comprise gut permeabilizing agents.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

b) Therapeutic Uses

Effective dosages and schedules for administering the compositionsdisclosed herein, including the peptides and multimers thereof disclosedherein, may be determined empirically, and making such determinations iswithin the skill in the art. The dosage ranges for the administration ofthe compositions are those large enough to produce the desired effect inwhich the symptoms/disorder is affected. The dosage should not be solarge as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage will vary with the age, condition, sex and extent of the diseasein the patient, route of administration, or whether other drugs areincluded in the regimen, and can be determined by one of skill in theart. The dosage can be adjusted by the individual physician in the eventof any counterindications. Dosage can vary, and can be administered inone or more dose administrations daily, for one or several days.Guidance can be found in the literature for appropriate dosages forgiven classes of pharmaceutical products, particularly for D-peptides.Examples of such guidance can be found throughout the literature. Forexample, the peptide FUZEON®, which has been FDA approved, can act as aguide for the dosages required for the peptides disclosed herein. In oneembodiment, the typical daily dosage of the peptides or multimersthereof used alone might range from about 1 μg/kg to up to 100 mg/kg ofbody weight or more per day, depending on the factors mentioned above.Furthermore, the peptides disclosed herein can be administered severaltimes daily, daily, weekly, monthly, or yearly, depending on thecondition of the subject, other modes of therapy, etc. One of skill inthe art could readily ascertain an appropriate dosing schedule.

Following administration of a disclosed composition, such as a peptidefor treating, inhibiting, or preventing a viral infection, such as HIV,the efficacy of the peptide or multimer thereof can be assessed invarious ways well known to the skilled practitioner. For instance, oneof ordinary skill in the art will understand that a composition, such asa D-peptide, disclosed herein is efficacious in treating or inhibiting aviral infection in a subject by observing that the composition inhibitsviral entry. Efficacy of the administration of the disclosed compositionmay also be determined by measuring the number of uninfected cells inthe infected subject. A treatment that inhibits an initial or furtherdecrease in uninfected cells in a subject or patient, or that result inan increase in the number of uninfected cells in, for example, theHIV-positive subject, is an efficacious treatment. The efficacy of aprophylactic treatment (i.e., preventative agent) can also be evaluatedusing indirect measures of infection, such as CD4+ cell counts, levelsof anti-virus antibodies, and PCR to detect viral RNA levels.

The compositions that inhibit viral entry, i.e., microbicides, disclosedherein may be administered prophylactically to patients or subjects whoare at risk for being exposed to a virus such as HIV or who have beennewly exposed to HIV. In subjects who have been newly exposed to a virussuch as HIV but who have not yet displayed the presence of the virus (asmeasured by PCR or other assays for detecting the virus) in blood orother body fluid, treatment with a peptide or multimer thereof includesadministering a therapeutically effective dose of a composition, apeptide or multimer as described herein to the subject such that theability of the virus to infect cells is partially or completelyinhibited.

The disclosed peptides can be used to inhibit viral entry by inhibitingviral transmembrane protein. The term “inhibit viral transmembraneprotein” refers to a reduction in the number of viral particles that arecapable of entering a cell. It can mean complete inhibition, in otherwords no viral particles are capable of entering a cell, or it can meana partial inhibition, meaning that in a given system there is areduction in the number of viral particles capable of entering a cellwhen compared with a non-treated system, or a control. There can be a 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or 100% reduction in the number of viralparticles that are capable of entering a cell, or any amount greater,less, or in between these amounts. Additionally, to “inhibit viralentry” means to reduce fusion and entry of virions into a cell.

3. Chips and Micro Arrays

Disclosed are chips where at least one address is the sequences or partof the sequences set forth in any of the peptide sequences disclosedherein.

Also disclosed are chips where at least one address is a variant of thesequences or part of the sequences set forth in any of the peptidesequences disclosed herein.

4. Computer Readable Mediums

It is understood that the disclosed peptides can be represented as asequence consisting of the amino acids. There are a variety of ways todisplay these sequences, for example the amino acid valine can berepresented by Val or V. Those of skill in the art understand how todisplay and express any peptide sequence in any of the variety of waysthat exist, each of which is considered herein disclosed. Specificallycontemplated herein is the display of these sequences on computerreadable mediums, such as, commercially available floppy disks, tapes,chips, hard drives, compact disks, and video disks, or other computerreadable mediums. Also disclosed are the binary code representations ofthe disclosed sequences. Those of skill in the art understand whatcomputer readable mediums. Thus, computer readable mediums on which thepeptide sequences are recorded, stored, or saved.

5. Compositions Identified by Screening with Disclosed Compositions

a) Combinatorial Chemistry

The disclosed peptides can be used as targets for any combinatorialtechnique to identify molecules or macromolecular molecules thatinteract with the disclosed compositions in a desired way. The peptidesand related molecules disclosed herein can be used as targets for thecombinatorial approaches. Also disclosed are the compositions that areidentified through combinatorial techniques or screening techniques inwhich the compositions disclosed in SEQ ID NOS: 1-36 for example, orportions thereof, are used as the template in a combinatorial orscreening protocol.

It is understood that when using the disclosed compositions incombinatorial techniques or screening methods, molecules, such asmacromolecular molecules, will be identified that have particulardesired properties such as inhibition or stimulation of gp41interactions. The molecules identified and isolated when using thedisclosed compositions, such as other peptides, are also disclosed.Thus, the products produced using the combinatorial or screeningapproaches that involve the disclosed compositions, such as peptides,are also considered herein disclosed.

b) Computer Assisted Drug Design

The disclosed peptides and multimers thereof can be used as targets forany molecular modeling technique to identify either the structure of thedisclosed peptides or multimers or to identify potential or actualmolecules, such as small molecules, which interact in a desired way withthe disclosed compositions. The peptides and related molecules disclosedherein can be used as targets in any molecular modeling program orapproach.

It is understood that when using the disclosed compositions in modelingtechniques, molecules, such as macromolecular molecules, will beidentified that have particular desired properties such as viralinhibition. The molecules identified and isolated when using thedisclosed compositions, such as peptides and multimers thereof, are alsodisclosed. Thus, the products produced using the molecular modelingapproaches that involve the disclosed compositions are also consideredherein disclosed.

Generally, one way to isolate molecules that bind a molecule of choiceis through rational design. This is achieved through structuralinformation and computer modeling. Computer modeling technology allowsvisualization of the three-dimensional atomic structure of a selectedmolecule and the rational design of new compounds that will interactwith the molecule. The three-dimensional construct typically depends ondata from x-ray crystallographic or NMR analysis of the selectedmolecule. Molecular dynamics simulations require force field data. Thecomputer graphics systems enable prediction of how a new compound willlink to the target molecule and allow experimental manipulation of thestructures of the compound and target molecule to perfect bindingspecificity. Prediction of what the molecule-compound interaction willbe when small changes are made in one or both requires molecularmechanics software and computationally intensive computers, usuallycoupled with user-friendly, menu-driven interfaces between the moleculardesign program and the user.

Examples of molecular modeling systems are the CHARMm and QUANTAprograms, Polygen Corporation, Waltham, Mass. CHARMm performs the energyminimization and molecular dynamics functions. QUANTA performs theconstruction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive withspecific proteins, such as Rotivinen, et al., 1988 Acta PharmaceuticaFennica 97, 159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988);McKinaly and Rossmann, 1989 Annu. Rev. Pharmacol. Toxiciol. 29, 111-122;Perry and Davies, QSAR: Quantitative Structure-Activity Relationships inDrug Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989Proc. R. Soc. Lond. 236, 125-140 and 141-162; and, with respect to amodel enzyme for nucleic acid components, Askew, et al., 1989 J. Am.Chem. Soc. 111, 1082-1090. Other computer programs that screen andgraphically depict chemicals are available from companies such asBioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario,Canada, and Hypercube, Inc., Cambridge, Ontario.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins.

6. Kits

Disclosed herein are kits that are drawn to reagents that can be used inpracticing the methods disclosed herein. The kits can include anyreagent or combination of reagent discussed herein or that would beunderstood to be required or beneficial in the practice of the disclosedmethods. For example, the kits could include a pharmaceuticalcomposition comprising a peptide or multimer thereof as disclosedherein. For example, disclosed is a kit for treating HIV, comprising apharmaceutical composition comprising a peptide or multimer thereof asdisclosed herein.

7. Compositions with Similar Functions

It is understood that the peptides disclosed herein have certainfunctions, such as inhibiting viral entry. Disclosed herein are certainstructural requirements for performing the disclosed functions, and itis understood that there are a variety of structures which can performthe same function which are related to the disclosed structures, andthat these structures will ultimately achieve the same result, forexample inhibiting viral entry.

C. Methods of Making the Compositions

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted.

1. Peptide Synthesis

The peptides disclosed herein can be linked, for example, by disulfidecrosslinks. For example, the D-peptides disclosed herein have two Cysresidues connected by a disulfide bond, which circularizes the peptideand creates a more compact and structured peptide. This disulfide isknown to have enhanced antiviral properties. There are many alternativemethods for circularizing peptides known to those of skill in the art.For example, a peptide can be circularized using lactam or otherchemical bridges, PEG or other chemical crosslinkers, peptide ligation,or diselenide bonds (between selenocysteines).

Two or more peptides or polypeptides can also be linked together byprotein chemistry techniques. For example, peptides or polypeptides canbe chemically synthesized using currently available laboratory equipmentusing either Fmoc (9fluorenylmethyloxycarbonyl) or Boc (tertbutyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City,Calif.). One skilled in the art can readily appreciate that a peptide orpolypeptide corresponding to the disclosed proteins, for example, can besynthesized by standard chemical reactions. For example, a peptide orpolypeptide can be synthesized and not cleaved from its synthesis resinwhereas the other fragment of a peptide or protein can be synthesizedand subsequently cleaved from the resin, thereby exposing a terminalgroup which is functionally blocked on the other fragment. By peptidecondensation reactions, these two fragments can be covalently joined viaa peptide bond at their carboxyl and amino termini, respectively, toform an antibody, or fragment thereof. (Grant G A (1992) SyntheticPeptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky Mand Trost B., Ed. (1993) Principles of Peptide Synthesis. SpringerVerlagInc., NY (which is herein incorporated by reference at least formaterial related to peptide synthesis). Once isolated, these independentpeptides or polypeptides may be linked to form a peptide or fragmentthereof via similar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen L etal., Biochemistry, 30:4151 (1991)). Alternatively, native chemicalligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al.Synthesis of Proteins by Native Chemical Ligation. Science, 266:776779(1994)). The first step is the chemoselective reaction of an unprotectedsynthetic peptidethioester with another unprotected peptide segmentcontaining an aminoterminal Cys residue to give a thioester linkedintermediate as the initial covalent product. Without a change in thereaction conditions, this intermediate undergoes spontaneous, rapidintramolecular reaction to form a native peptide bond at the ligationsite (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; ClarkLewis I etal., J. Biol. Chem., 269:16075 (1994); ClarkLewis I et al.,Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry33:6623-30 (1994)).

Alternatively, unprotected peptide segments are chemically linked wherethe bond formed between the peptide segments as a result of the chemicalligation is an unnatural (nonpeptide) bond (Schnolzer, M et al. Science,256:221 (1992)). This technique has been used to synthesize analogs ofprotein domains as well as large amounts of relatively pure proteinswith full biological activity (deLisle Milton R C et al., Techniques inProtein Chemistry IV. Academic Press, New York, pp. 257267 (1992)).

Mirror-image phage display can be used to discover D-peptides that bindto the N-trimer pocket and inhibit HIV-1 entry with modest potency. Forexample, in using mirror-image phage display to screen for D-peptides, afirst D-peptide can be synthesized from the first L-peptide from a HIVglycoprotein. The first L-peptide can be a naturally occurring L-peptideor can be a chimera of designed peptide sequences and natural peptidesequences. The methods can further comprise screening for a secondL-peptide that specifically binds to the first D-peptide; then, a secondD-peptide that is the mirror image of the second L-peptide can besynthesized. In one aspect of the D-peptide screening methods describedherein, an N-trimer target can first be synthesized with D-amino acids,creating the mirror image of the natural L-N-trimer target. TheD-N-trimer target can be used in standard peptide-based screens such asphage display, ribosome display, and/or CIS display to identifyL-peptides that bind to the D-N-trimer. The identified L-peptides canthen be synthesized with D-amino acids. By the law of symmetry, theresulting D-peptides bind the natural L-N-trimer, and will thus targetthe N-trimer region of the HIV prehairpin intermediate, thereby treatingor inhibiting HIV infection. This screening method is also described inSchumacher, et al., Identification of D-peptide ligands throughmirror-image phage display, Science, 1996 Mar. 29; 271(5257):1854-7,which is hereby incorporated in its entirety by this reference.

D. Methods of Using the Compositions

1. Methods of Using the Compositions as Research Tools

Disclosed herein are methods for evaluating the ability of a compositioncomprising a peptide of less than 10 core residues in length for itsability to inhibit viral entry into a cell comprising: incubating thecomposition and a cell under conditions sufficient to allow thecomponents to interact; contacting the components with a virus; andevaluating the ability of the composition to inhibit viral entry intothe cell. The peptide can comprise less than 7, 8, 9, or 10 core aminoacid residues. The peptide can be present as a multimer, as disclosedabove. The composition can inhibit viral entry by interacting with aviral transmembrane protein, such as HIV gp41. The peptide can be aD-peptide. Furthermore, evaluating the ability of the composition toinhibit viral entry can be by detection of a reporter means. Examples ofsuch reporter means include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, a chemiluminescentcompound, a metal chelator, or an enzyme. Evaluating the ability of thecomposition to inhibit viral entry into the cell can be done byevaluating the ability of the composition to be displaced from itsbinding site (the gp41 N-trimer pocket) by other compounds (e.g.,peptides, small molecules, nucleic acids, natural products). By“displaced” is meant that the composition is inhibited from binding, oris disrupted from its interaction with the binding site. This can occurat 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, or 100% displacement of the test compositions from the bindingsite.

The ability of the composition to inhibit viral entry can be measuredusing viral entry assays or cell-cell fusion assays. Viral entry assaysare known to those in the art, as are cell-cell fusion assays. One canuse a displacement assay comprising other compounds that can displacethe test composition from the binding site. Examples include, but arenot limited to, peptides, small molecules, nucleic acids, or naturalproducts. Such displacement assays are known to those of skill in theart.

Also disclosed is a method of identifying a multimer with increasedaffinity for an N-trimer molecule when compared with the affinity of oneof a single peptide, comprising: incubating the multimer and an N-trimermolecule; measuring the affinity of the multimer for the N-trimermolecule; and comparing the affinity of the multimer for the N-trimermolecule with the affinity for the N-trimer molecule of a singlepeptide.

Further disclosed is a method of identifying a multimer with enhancedantiviral activity for an N-trimer molecule when compared with theantiviral activity of one a single peptide, comprising: incubating themultimer with a cell; contacting the components of step (a) with avirus; measuring the antiviral activity of the multimer; and comparingthe antiviral activity of the multimer with the antiviral activity of asingle peptide. The single peptide can be identical to one of thecomponents of the multimer, or can be different. The multimer cancomprise at least one peptide which interacts with the N-trimer pocketof viral gp41.

Inhibition of complex formation of gp41 can be assessed by determiningthe extent to which binding of the two members of the complex occurs,such as by means of a fluorescence assay (e.g., FRET), in which C34 andN36 are each labeled by a member of a pair of donor-acceptor moleculesor one end of one of the peptides (e.g., the N-terminus of C34) islabeled with one member of such a pair (EDANS) and the naturalfluorophore tryptophan, present in the N36 peptide, is the other memberof the donor/acceptor pair. Binding of the C34 and N36 is assessed bythe extent to which light emission (FRET) occurs from the acceptor modeland/or the wavelength spectrum of the light emitted is altered.Prevention of binding by the candidate drug alters the extent to whichlight is emitted and/or prevents the shift in wavelength that wouldoccur if binding of C34 and N36 occurred. Alternatively, C34 can belabeled with a detectable label, such as a radiolabel (e.g., bysynthesizing a variant C34 with a kinase recognition site that can belabeled with a kinase and radioactive ATP). The radiolabeled C34 and thecandidate drug are combined with N36 immobilized to, for example, asolid surface (e.g., a bead or a plastic well), thus producing a testsample. The extent to which binding of labeled C34 with immobilized N36occurs is determined and compared with the extent to which binding oflabeled C34 to immobilized N36 occurs under the same conditions to whichthe test sample is subjected, but in the absence of the candidate drug(in a control sample). Typically, this assessment is carried out afterthe sample has been maintained for sufficient time and under appropriateconditions for C34/N36 binding to occur and a subsequent wash to removeany unbound C34 and candidate drug. If binding occurs in the test sampleto a lesser extent than in the control sample, as evidenced by lessradiolabel bound to the immobilized N36 in the test sample than in thecontrol sample, the candidate drug is an inhibitor of binding of C34 andN36. Alternatively, the label or tag on C34 can be a member of a bindingpair, the other member of which is used to detect binding to N36. Forexample, C34 can be tagged with biotin (through standard solid-statepeptide synthesis, for example) and combined with N36, which can be insolution or bound to a solid surface, such as a bead, well orflat/planar surface, along with the candidate drug (test sample) or inthe absence or the candidate drug (control sample). Binding of C34 toN36 is assessed by detecting the presence of biotin associated with N36,such as through the use of labeled streptavidin (e.g., streptavidin—HRP,streptavidin—AP or iodinated streptavidin), which binds the biotin onC34 and is then itself detected through its label. If binding occursless in the presence of the candidate drug (in the test sample) than inthe absence of the candidate drug (in the control sample), as indicatedby the presence of less biotin detected on N36 in the test sample thanin the control sample, the candidate drug is an inhibitor of C34/N36binding. The candidate drugs can be obtained, for example, from alibrary of synthetic organic compounds or random peptide sequences,which can be generated synthetically or through recombinant technology.

In a similar fashion, the ability of a candidate drug to disrupt C34/N36binding can be assessed, to identify inhibitors of C34/N36 and, thus, ofHIV infection. In this embodiment, preformed C34/N36 complex is combinedwith a candidate drug, which is to be assessed for its ability todisrupt the complex, thus producing a test sample. The control sample isthe same as the test sample, except that the control sample does notcontain the candidate drug; it is treated in the same manner as the testsample. If C34/N36 binding is disrupted in the presence of the candidatedrug and not in the control sample or if disruption of the complexoccurs to a greater extent in the test sample than in the controlsample, the candidate drug is an inhibitor (disrupter) of C34/N36.Detection of disruption of binding can be carried out as described abovefor detection of/prevention of/interference with binding of C34/N36(e.g., by FRET or a fluorescence assay, by detecting a radiolabel orother detectable label, such as biotin.)

In another embodiment, the current disclosure relates to a method ofidentifying a drug that binds the N-helix coiled-coil cavity of HIVgp41. Here, too, the assay is based on assessing loss or decrease inbinding, but unlike the C34/N36 complex assay described above, which isa more general assay in that it covers or detects interaction with anyportion of the groove formed by the N-helical region of HIV gp41, thisembodiment focuses on the HIV gp41 hydrophobic pocket (the N-helixcoiled-coil cavity). In this embodiment, the method comprises combininga candidate drug to be assessed for its ability to bind the N-helixcoiled-coil cavity of HIV gp41 with a fusion protein that comprises atrimeric version of the coiled-coil region of a protein and a sufficientportion of the N-peptide of HIV gp41 to include the HIV gp41 cavity,under conditions appropriate for presentation of the HIV gp41 cavity forbinding by a peptide or other molecule and determining (e.g., in a highthroughput screen) whether the candidate drug binds the fusion protein.If binding occurs, the candidate drug is a “hit” that may be a drug thatbinds the N-helix coiled-coil cavity of HIV gp41. If binding occurs, thecandidate drug has bound the N-helix coiled coil and it can bedetermined if it binds to the coiled-coil cavity. Such “hits” can thenbe screened in secondary assays, such as the cell/cell fusion assay andHIV infectivity assay to determine if the candidate drug is a drug.Alternatively, or in addition, such “hits” can be assessed further byuse of a counterscreen with other fusion proteins (or peptides), towhich pocket-binding molecules will not bind.

In a further embodiment, a competitive assay is carried out. In thisembodiment, a peptide or protein that binds the N-helix coiled-coilcavity of HIV gp41 is combined with the candidate drug and the fusionprotein and whether the candidate drug binds the HIV gp41 cavity isdetermined in the presence of the peptide that binds the N-helix coiledcavity of HIV gp41. If the candidate drug binds the fusion protein, itis a drug that binds the HIV gp41 cavity. For example, a fusion proteinwhich comprises a trimeric version of the coiled-coil region of GCN4 andthe C-terminus of the N peptide of HIV gp41 that includes the N-helixcoiled-coil cavity (IQN17) is combined with a “reference” D-peptide(e.g., any of the D-peptides described herein or variants thereof) thatbinds the N-helix coiled-coil cavity and a candidate drug to be assessedfor its ability to bind the N-helix coiled-coil cavity of HIV gp41, thusproducing a test sample, which is maintained under conditionsappropriate for binding of the D-peptide to bind to the cavity. Acontrol sample, which includes the same components as the test sample,except for the candidate drug, and is handled in the same manner as thetest sample, is also assessed. In both samples, binding of the referenceD-peptide is assessed. If binding of the reference D-peptide occurs to alesser extent in the presence of the candidate drug (in the test sample)than in its absence (in the control sample), the candidate drug is adrug that binds the N-helix coiled-coil cavity of HIV gp41. Detection ofbinding is assessed, for example, in a similar manner as described abovefor the C34/N36 embodiment of the disclosure. For example, the D-peptideis labeled with a detectable label, such as a radiolabel or a firstmember of a binding pair (e.g., biotin), and the extent to which theN-helix coiled-coil cavity bears the label (after the samples have beenmaintained under conditions appropriate for binding of the referenceD-peptide to the cavity) is determined. In the case in whichradiolabeling is used, the extent to which the fusion protein bears theradiolabel is assessed in the test sample and compared with the extentto which the fusion protein bears the radiolabel in the control sample.If the detectable label is a first member of a binding pair (e.g.biotin), the second member of the pair (a binding partner) is added tothe samples in order to detect the extent to which the fusion protein isbound by the reference D-peptide. This can be done directly orindirectly (e.g., by adding a molecule, such as an antibody or othermoiety which binds the second member of the binding pair). Less of thelabel will be present on the fusion protein (N-helix coiled-coil cavity)if the candidate drug has inhibited (totally or partially) binding ofthe D-peptide to the cavity. If binding occurs to a lesser extent in thetest sample (in the presence of the candidate drug) than in the controlsample (in the absence of the candidate drug), then the candidate drugis a drug that binds the N-helix coiled-coil cavity of HIV gp41.

IQN17, or a variant thereof, in the D-enantiomer, is useful to identifymolecules or compounds which are members of a library or collection andbind the N-helix coiled-coil of gp41. For example, a library orcollection of molecules or compounds, such as a phage display library,can be screened with IQN17 in the D-enantiomer to identify members thatbind the pocket. This has been carried out successfully, as describedherein. The mirror image of IQN17, or a variant thereof, is used as thetarget molecule. As used herein, the terms “D-enantiomer of apolypeptide” and “D-peptide” refer to the exact mirror image of themolecule in the natural handedness. Thus, for amino acid residues thatcontain a second chiral center, such as Ile and Thr, the exact mirrorimage of the naturally-occurring amino acid residue is used to createthe D version of the polypeptide. Also as used herein, the terms“D-amino acids” and “L-amino acids” are both meant to include thenon-chiral amino acid glycine. D-IQN17 can be immobilized to a solidsurface, such as by addition of one member of a binding pair (e.g.,biotin) to it and addition of the other member of the pair (e.g.,streptavidin) to the solid surface. Binding of the two members resultsin immobilization of D-IQN17 on the solid surface, such as for phagepanning. A linker which is an enzyme recognition site (e.g., an aminoacid linker such as Gly-Lys-Gly, in which an L-lysine residue is used)can be placed between the D-IQN17 sequence and the binding pair member(between the biotin and D-IQN17) to provide an enzyme recognition site(here, a trypsin recognition site), so that bound phage can be eluted bya trypsin digestion, rather than by non-specific elution, such as acidaddition. The phage display library can be a library of L-amino acidpeptides of any appropriate length fused to an appropriate phage gene.In one embodiment, it is a phage display library of L-amino acidpeptides fused to the gIII gene of M13 phage. The peptides, in oneembodiment, comprise 10 randomly encoded amino acid residues flanked byeither a cysteine or a serine on both sides. Typically, several roundsof panning are carried out. D-IQN17-specific binding phage areidentified. Phage that bind only the gp41 region of D-IQN17 can beidentified by post-panning assessment, such as by screening againstwells that lack the antigen and then further testing against a panel ofmolecules. For example, specific pocket-binding phage include those thatbind DIQN17 but not D-GCN4-pIQI (with the same three surface mutationsas in IQN17) or a version of D-IQN17 with a point mutation in thehydrophobic pocket, D-IQN17(G39W), in which glycine 39 is mutated totryptophan, resulting in a large protrusion into the pocket. D-peptidesidentified in this manner can be assessed for their ability to inhibitHIV gp41, using known assays, such as the cell/cell fusion assay and HIVinfectivity assay. The mirror-image phage display method describedherein has demonstrated the value of IQN17 and IQN17(G39W), and theirD-enantiomers in identifying inhibitors of HIV-1 entry that bind thegp41 pocket. Of nine specific pocket-binding phage sequences identified(phage that bind to D-IQN17 but not to D-IQN17(G39W), eight contain aconsensus EWXWL sequence and inhibit HIV-1 gp41-induced syncytiaformation when tested as D-peptides. The ninth peptide was toxic tocells and was not investigated further.

The D-versions of IQN17 and IQN17(G39W) can be used in a similar mannerwith other biologically encoded libraries, to discover otherpocket-binding molecules that are not subject to enzymatic degradationby natural enzymes. For example, other phage-display libraries can beused to identify new D-peptide inhibitors (e.g., with a different numberof residues between the flanking Cys residues and/or with randomlyencoded amino acid residues outside the regions flanked by cysteineresidues and/or with more than two cysteine residues). Strategies forencoding peptide libraries without phage (e.g., in which the encodingmRNA is attached to the peptide) can be used to identify D-peptideinhibitors. RNA or DNA libraries can be used (e.g., with SELEX methods)to identify L-ribose- or L-deoxyribose-based RNA or DNA aptamers,respectively, that bind to the hydrophobic pocket and are not substratesfor natural nucleases (see e.g., Williams et al., PNAS, 74:11285(1997)).

Although the versions of IQN17 and IQN17(G39W) of natural L-handednesscan also be used in similar manner with biologically encoded libraries,the most likely applications will be with other, non-biologicallyencoded libraries. For example, chemical combinatorial libraries onbeads (of the one-bead, one-compound variety) can be screened withlabeled IQN17 (e.g., radioactive or with a chromophore) to identifybeads containing molecules that bind to IQN17. In this example,IQN17(G39W) can be used as a counterscreen to determine if the moleculeson the bead bind to the pocket of IQN17. (If they bind to IQN17(G39W),then they are not likely to be pocket-binding molecules.) As anotherexample, beads to which IQN17 had been previously attached can beincubated with a mixture of potential pocket-binding molecules (e.g., amixture of chemicals, or a natural product extract). IQN17 (bound to thebeads) can then be separated from the mixture, washed, and thensubjected to conditions (e.g., organic solvent, low pH, hightemperature) that elute molecules bound to the IQN17 on the beads. Theeluted molecules (i.e., potential pocket-binding molecules) could beidentified by analytical chemistry methods (e.g., HPLC, massspectrometry). A counterscreen with IQN17(G39W) is useful to help toidentify true pocket-binding molecules.

Drugs identified by the methods described above are then further testedfor their ability to inhibit (totally or partially) HIV gp41 function(membrane fusion) and, thus entry into cells, using further in vitroassays, such as the syncytium assays and/or infectivity assays describedherein or others known to those of skill in the art, and/or in vivoassays in appropriate animal models or in humans.

One embodiment of the present disclosure is a method of identifying adrug that binds the N-helix coiled-coil of HIV gp41, particularly theN-helix coiled-coil pocket. The method comprises combining a candidatedrug to be assessed for its ability to bind the N-helix coiled-coilpocket of HIV gp41 and peptide which comprises a soluble, trimericcoiled-coil and a sufficient portion of the N-peptide of HIV gp41 toinclude the HIV gp41 pocket, under conditions appropriate forpresentation of the HIV gp41 pocket for binding by a molecule orcompound (e.g., a drug) and determining whether the candidate drug bindsthe HIV gp41 pocket. If binding of the candidate drug with the HIV gp41pocket occurs, the candidate drug is a drug which binds the N-helixcoiled-coil pocket of HIV gp41. Optionally, binding of the candidatedrug can be assessed in the assay as described above, except that apeptide that binds the N-helix coiled-coil pocket (a peptide previouslyidentified as one which binds the pocket) is combined with the candidatedrug and the peptide. In this competitive assay, binding of thecandidate drug to the N-helix coiled-coil pocket is assessed in thepresence of a known binding moiety (a molecule or compound which bindsthe pocket). If binding of the candidate drug occurs in the presence ofthe known binding moiety, the candidate drug is a drug which binds theN-helix coiled-coil pocket with sufficient affinity to successfullycompete with the known binding moiety. The fusion protein used in thisembodiment comprises a soluble, trimeric version of a coiled-coil, suchas a soluble, trimeric version of the coiled-coil region of a proteinand a sufficient portion of the N-peptide of HIV gp41 to include the HIVgp41 cavity. Alternatively, a variant of the HIV gp41 sequence presentherein, a sequence from another strain of the human virus (e.g., HIV-2)or a sequence from another species (e.g., SIV, feline immunodeficiencyvirus, Visna virus (M. Singh et al., J. Mol. Biol., 290:1031 (1999)) canbe used in the fusion protein or soluble model. The fusion protein cancomprise a soluble, trimeric version of the coiled-coil of any protein,provided that when it is in the fusion protein with the HIV component,the HIV cavity is presented in such a manner that it is available forbinding. It can be, for example, that of GCN4-pIQI, GCN4-pII, MoloneyMurine Leukemia Virus (Mo-MLV) or the ABC heterotrimer. In oneembodiment, the fusion protein is IQN17 in the D-form. In anotherembodiment, the fusion protein is IQN17 in the natural L-handedness.

In the competitive assay format, any peptide known to bind the N-helixcoiled-coil cavity can be used as the known binding moiety. For example,any of the peptides described herein or a variant or portion thereof canbe used. Also, any non-peptide pocket-binding molecule can be used inthe competitive assay format. The competitive assay can be performed insolution, on a bead, or on a solid surface.

In one embodiment, the candidate drug is detectably labeled and bindingof the candidate drug to the HIV gp41 N-helix coiled-coil is determinedby detecting the presence of the detectable label on the HIV gp41N-helix coiled-coil (as a result of binding of the labeled candidatedrug to the N-helix coiled-coil). Detection of the label on the helixcoiled-coil pocket of the soluble model is indicative of binding of thecandidate drug to the N-helix coiled-coil pocket and demonstrates thatthe candidate drug is a drug which binds the N-helix coiled-coil pocket.If the labeled candidate drug is detected on the fusion protein, thecandidate drug is a drug which binds the N-helix coiled-coil cavity.

In another embodiment of the method of identifying a drug that binds theN-helix coiled-coil pocket of the HIV gp41, a soluble model thatpresents the pocket in such a manner that it is available for binding bya drug is combined with a candidate drug and whether binding of thecandidate drug with the N-helix coiled-coil of the soluble model occursis determined. If binding occurs, the candidate drug is a drug whichbinds the pocket. Here, too, a competitive assay format can be used. Thecomponents of the competition assay (e.g., IQN17 and a D-peptide) can belabeled, with any of a variety of detectable labels, includingfluorophore/quencher combinations. The candidate drug can be labeled, asdescribed above, with any of a variety of detectable labels. Thecomponents of the soluble model (fusion protein) used in this embodimentand the competing moiety which is used in a competitive assay format canalso be as described above.

The present disclosure also relates to a method of producing a drug thatbinds the N-helix coiled-coil pocket of HIV gp41. In one embodiment, themethod is carried out as follows: A soluble model that presents theN-helix coiled-coil pocket of HIV gp41 or a fusion protein whichcomprises a soluble, trimeric coiled-coil is combined with a candidatedrug to be assessed for its ability to bind the N-helix coiled-coilpocket of HIV gp41 and inhibit entry into cells, under conditionsappropriate for presentation of the HIV gp41 pocket for binding by adrug. Whether the candidate drug binds the HIV gp41 pocket isdetermined, wherein if binding of the candidate drug to the N-helixcoiled-coil pocket of HIV gp41 occurs, the candidate drug is a drugwhich binds the N-helix coiled-coil cavity of HIV gp41. In thisembodiment, the fusion protein comprises a soluble, trimeric coiled-coiland a sufficient portion of the N-peptide of HIV gp41 to include the HIVgp41 N-helix coiled-coil pocket IQN17, described herein, can be used inthis method; the D enantiomer of IQN17 can also be used (e.g., inmirror-image phage applications). The ability of the drug produced toinhibit HIV entry into cells is assessed, for example, in a syncytiumassay and/or an infectivity assay, as described herein. It can befurther assessed in an appropriate animal model or in humans.

Also disclosed herein is a method of producing a drug that binds theN-helix coiled-coil pocket of HIV gp41. The method comprises: producingor obtaining a soluble model of the N-helix coiled-coil pocket of HIVgp41; combining a candidate drug (a molecule or compound) to be assessedfor its ability to bind the N-helix coiled-coil pocket of HIV gp41 andthe soluble model of the N-helix coiled-coil pocket of HIV gp41 anddetermining whether the candidate drug binds the N-helix coiled-coilpocket of HIV gp41. If the candidate drug binds the N-helix coiled-coilpocket of HIV gp41, the candidate drug is a drug which binds the N-helixcoiled-coil pocket of HIV gp41; as a result, a drug which binds theN-helix coiled-coil cavity of HIV gp41 is produced. The fusion proteinused in this embodiment is described herein and can be, for example,IQN17, the D enantiomer of IQN17, or variants thereof. Alternatively, adrug that binds the N-helix coiled-coil pocket of HIV gp41 and inhibitsentry of HIV into cells can be produced by a method comprising:producing or obtaining a soluble model of the N-helix coiled-coil pocketof HIV gp41, as described herein; combining the soluble model and acandidate drug to be assessed for its ability to bind the N-helixcoiled-coil pocket of HIV gp41; determining whether the candidate drugbinds the N-helix coiled-coil pocket of the soluble model (fusionprotein), wherein if binding occurs, the candidate drug is a drug whichbinds the N-helix coiled-coil of HIV gp41; and assessing the ability ofthe drug which binds the N-helix coiled-coil to inhibit HIV entry intocells, wherein if the drug inhibits HIV entry into cells, it is a drugwhich binds the N-helix coiled-coil pocket of HIV gp41 and inhibits HIVentry into cells. Its ability to inhibit HIV entry into cells can beassessed in vitro (e.g., in a syncytium assay, an infectivity assay) orin vivo (e.g. in an appropriate animal model or in humans). The solublemodel can be a peptide which comprises a soluble, trimeric coiled-coil,such as that of a protein and a sufficient portion of the N-peptide ofHIV gp41 to include the HIV gp41 pocket.

Drugs identified or produced by the methods described herein, as well asby other methods, which bind the N-helix coiled-coil pocket of HIV gp41and inhibit HIV entry into cells are also the subject of thisdisclosure.

Drugs identified or produced by the methods described herein, as well asby other methods, which bind to more than one N-helix coiled-coil pocketof HIV gp41 and inhibit HIV entry into cells are also the subject ofthis disclosure. Such drugs can be obtained, for example, by linking twoor more pocket-binding molecules (drugs) via an appropriate linker(e.g., a linker of amino acid residues or other chemical moieties) toincrease the effectiveness of inhibition. The pocket-binding moleculesthat are linked can be the same or different. Drugs identified orproduced by the methods described herein or by other methods which bindto the N-helix coiled-coil pocket of HIV gp41, in addition to binding toHIV gp120, CD4, CCR5, CXCR4, or a non-pocket region of HIV gp41 are alsothe subject of this disclosure.

Drugs which inhibit HIV gp41 can also be designed or improved withreference to the X-ray crystal structure of the complex IQN17 and2K-PIE1, which is presented herein. Alternatively, or in addition, drugswhich inhibit HIV gp41 can also be designed or improved with referenceto the X-ray crystal structure of free IQN17, presented herein.

Compounds and molecules (drugs) identified as described herein inhibit(partially or totally) entry of HIV into cells, and thus are usefultherapeutically in uninfected individuals (humans) and infectedindividuals (e.g., to prevent or reduce infection in an uninfectedindividual, to reduce or prevent further infection in an infectedindividual) and as research reagents both to study the mechanism ofgp41-induced membrane fusion and to assess the rate of viral clearanceby an individual and as reagents to discover or develop other compoundsand molecules (drugs) that inhibit entry of HIV into cells. D-peptidesdescribed herein (e.g., D10pep5, D10pep1) have been shown, using theinfectivity assay described herein, to inhibit infection of cells. OtherD-peptides can be similarly assessed for their ability to inhibitinfectivity.

As disclosed above, putative inhibitors can be identified usingFluorescence Resonance Energy Transfer (FRET) to quickly identifyinteractions. The underlying theory of the techniques is that when twomolecules are close in space, i.e., interacting at a level beyondbackground, a signal is produced or a signal can be quenched. Then, avariety of experiments can be performed, including, for example, addingin a putative inhibitor. If the inhibitor competes with the interactionbetween the two signaling molecules, the signals will be removed fromeach other in space, and this will cause a decrease or an increase inthe signal, depending on the type of signal used. This decrease orincreasing signal can be correlated to the presence or absence of theputative inhibitor. Any signaling means can be used. For example,disclosed are methods of identifying an inhibitor of the interactionbetween any two of the disclosed molecules comprising, contacting afirst molecule and a second molecule together in the presence of aputative inhibitor, wherein the first molecule or second moleculecomprises a fluorescence donor, wherein the first or second molecule,typically the molecule not comprising the donor, comprises afluorescence acceptor; and measuring Fluorescence Resonance EnergyTransfer (FRET), in the presence of the putative inhibitor and the inabsence of the putative inhibitor, wherein a decrease in FRET in thepresence of the putative inhibitor as compared to FRET measurement inits absence indicates the putative inhibitor inhibits binding betweenthe two molecules. This type of method can be performed with a cellsystem as well.

There are a number of methods for isolating proteins which either havede novo activity or a modified activity. For example, phage displaylibraries have been used to isolate numerous peptides that interact witha specific target. (See for example, U.S. Pat. Nos. 6,031,071;5,824,520; 5,596,079; and 5,565,332 which are herein incorporated byreference at least for their material related to phage display andmethods relate to combinatorial chemistry)

Using methodology well known to those of skill in the art, incombination with various combinatorial libraries, one can isolate andcharacterize those small molecules which bind to or interact with thedesired target. The relative binding affinity of these compounds can becompared and optimum compounds identified using competitive bindingstudies, which are well known to those of skill in the art.

Techniques for making combinatorial libraries and screeningcombinatorial libraries to isolate molecules which bind a desired targetare well known to those of skill in the art. Representative techniquesand methods can be found in but are not limited to U.S. Pat. Nos.5,084,824, 5,288,514, 5,449,754, 5,506,337, 5,539,083, 5,545,568,5,556,762, 5,565,324, 5,565,332, 5,573,905, 5,618,825, 5,619,680,5,627,210, 5,646,285, 5,663,046, 5,670,326, 5,677,195, 5,683,899,5,688,696, 5,688,997, 5,698,685, 5,712,146, 5,721,099, 5,723,598,5,741,713, 5,792,431, 5,807,683, 5,807,754, 5,821,130, 5,831,014,5,834,195, 5,834,318, 5,834,588, 5,840,500, 5,847,150, 5,856,107,5,856,496, 5,859,190, 5,864,010, 5,874,443, 5,877,214, 5,880,972,5,886,126, 5,886,127, 5,891,737, 5,916,899, 5,919,955, 5,925,527,5,939,268, 5,942,387, 5,945,070, 5,948,696, 5,958,702, 5,958,792,5,962,337, 5,965,719, 5,972,719, 5,976,894, 5,980,704, 5,985,356,5,999,086, 6,001,579, 6,004,617, 6,008,321, 6,017,768, 6,025,371,6,030,917, 6,040,193, 6,045,671, 6,045,755, 6,060,596, and 6,061,636.

Combinatorial libraries can be made from a wide array of molecules usinga number of different synthetic techniques. For example, librariescontaining fused 2,4-pyrimidinediones (U.S. Pat. No. 6,025,371)dihydrobenzopyrans (U.S. Pat. Nos. 6,017,768 and 5,821,130), amidealcohols (U.S. Pat. No. 5,976,894), hydroxy-amino acid amides (U.S. Pat.No. 5,972,719) carbohydrates (U.S. Pat. No. 5,965,719),1,4-benzodiazepin-2,5-diones (U.S. Pat. No. 5,962,337), cyclics (U.S.Pat. No. 5,958,792), biaryl amino acid amides (U.S. Pat. No. 5,948,696),thiophenes (U.S. Pat. No. 5,942,387), tricyclic Tetrahydroquinolines(U.S. Pat. No. 5,925,527), benzofurans (U.S. Pat. No. 5,919,955),isoquinolines (U.S. Pat. No. 5,916,899), hydantoin and thiohydantoin(U.S. Pat. No. 5,859,190), indoles (U.S. Pat. No. 5,856,496),imidazol-pyrido-indole and imidazol-pyrido-benzothiophenes (U.S. Pat.No. 5,856,107) substituted 2-methylene2, 3-dihydrothiazoles (U.S. Pat.No. 5,847,150), quinolines (U.S. Pat. No. 5,840,500), PNA (U.S. Pat. No.5,831,014), containing tags (U.S. Pat. No. 5,721,099), polyketides (U.S.Pat. No. 5,712,146), morpholino-subunits (U.S. Pat. Nos. 5,698,685 and5,506,337), sulfamides (U.S. Pat. No. 5,618,825), and benzodiazepines(U.S. Pat. No. 5,288,514).

As used herein combinatorial methods and libraries included traditionalscreening methods and libraries as well as methods and libraries used initerative processes. The disclosed peptides can be used in a variety ofways as research tools. For example, the disclosed peptides, such as SEQID NOS: 1-22 can be used to study gp41, by for example acting asinhibitors of viral entry or of proper folding of the protein.

2. Methods of Inhibiting Viral Entry

Disclosed herein are methods for inhibition of transmission of a virusto a cell, or inhibiting viral entry, comprising exposing the virus tocompositions, peptides or multimers as disclosed herein, and therebyinhibiting transmission of the virus to the cell. The virus can be HIV.The peptides or multimers can be in a pharmaceutical composition. Alsodisclosed are methods of administering a pharmaceutical compositiondescribed herein.

In certain embodiments, the methods disclosed herein for inhibition oftransmission of a virus to a cell comprise administering a compositioncomprising at least one D-peptide linked to a potency-enhancing cargowith a polyethylene glycol (PEG) linker. In particular embodiments, themethods comprise administering a composition comprising at least oneD-peptide and a potency-enhancing cargo, wherein the at least oneD-peptide is at least one of SEQ ID NOS: 1-29, and wherein thepotency-enhancing cargo is at least one of a cholesterol, a fatty acid,and an alkane chain. In one such embodiment, the methods compriseadministering a composition comprising chol-PEG₂₄-PIE12.

In other embodiments, the methods disclosed herein for inhibition oftransmission of a virus to a cell comprise administering a compositioncomprising at least three D-peptides linked to a multimer scaffold,wherein the at least three D-peptides are linked to the multimerscaffold with a PEG linker. In certain such embodiments, the methodscomprise administering a composition comprising at least threeD-peptides linked to a multimer scaffold, wherein the at least threeD-peptides comprise at least one of SEQ ID NOS: 1-29. In one suchembodiment, the methods comprise administering a composition comprisingPEG₄-PIE12-trimer.

In some embodiments, the methods disclosed herein for inhibition oftransmission of a virus to a cell comprise administering a compositioncomprising at least three D-peptides and at least one potency-enhancingcargo linked to a multimer scaffold. In some particular embodiments, themethods comprise administering a composition comprising at least threeD-peptides and at least one potency-enhancing cargo linked to a multimerscaffold, wherein the multimer scaffold is a heterotetrameric scaffoldcomprising three NHS ester groups and a fourth orthogonal group. Instill other embodiments, the methods comprise administering acomposition comprising at least three D-peptides and at least onepotency-enhancing cargo linked to a multimer scaffold wherein the atleast one potency-enhancing group is linked to the multimer scaffold viaa fourth orthogonal group with a PEG linker. In some particularembodiments, the methods comprise administering a composition comprisingat least three D-peptides and at least one potency-enhancing cargolinked to a multimer scaffold wherein the at least one potency-enhancinggroup is a cholesterol, a fatty acid, or a alkane chain. In one suchembodiment, the methods comprise administering a composition comprisingat least one of chol-PEG₁₂-PIE12-trimer, chol-PEG₁₆-PIE12-trimer,chol-PEG₂₄-PIE12-trimer, chol-PEG₃₆-PIE12-timer,chol-PEG₅₇-PIE12-trimer, chol-PEG₁₃₂-PIE12-trimer, C8 fattyacid-PEG₂₄-PIE12-trimer, C16 fatty acid-PEG₂₄-PIE12-trimer, C18 fattyacid-PEG₂₄-PIE12-trimer, C8 alkane-PEG₂₄-PIE12-trimer, C16alkane-PEG₂₄-PIE12-trimer, and C18 alkane-PEG₂₄-PIE12-trimer.

The methods disclosed herein can be used in conjunction with other viraltherapies or antiviral agents. One of more of these antiviral agents canbe used, and they can be administered before, during, or after treatmentwith the compositions disclosed herein. For example, in ongoing therapy,the subject can be administered the compositions comprised hereinsimultaneously with other treatments, meaning they can be administeredabout 48 hours, 24 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour,30 minutes, 20 minutes, 10 minutes, 5 minutes, or one minute beforetreatment with the disclosed compositions. Other methods of treatmentcan also be administered before treatment with the compositionsdisclosed herein. By “before treatment” is meant that another form oftreatment was given and then stopped before the current therapy wasadministered, or could be given immediately before, then administeredagain afterwards. In this case, the other methods of antiviral therapycan be administered years, months, weeks, days, hours, or minutes inadvance. Other methods of treatment can also be administered aftertreatment with the compositions disclosed herein. By “after treatment”is meant that another form of treatment is administered after thecurrent therapy was administered, or could be given before, thenadministered again afterwards. This additional antiviral treatment couldbe given years, months, weeks, days, hours, or minutes after the currenttherapy is given.

The further antiviral agent or agents can be selected from the groupconsisting of a viral replication inhibitor, a viral protease inhibitor,a viral reverse transcriptase inhibitor, a viral entry inhibitor, aviral integrase inhibitor, a viral Rev inhibitor, a viral Tat inhibitor,a viral Nef inhibitor, a viral Vpr inhibitor, a viral Vpu inhibitor, anda viral Vif inhibitor.

Further examples of antiviral compounds include, but are not limited to,amantadine, rimantadine, zanamavir and oseltamavir (Tamiflu) for thetreatment of flu and its associated symptoms. Antiviral compounds usefulin the treatment of HIV include Combivir® (lamivudine-zidovudine),CRIXIVAN® (indinavir), EMTRIVA® (emtricitabine), EPIVIR® (lamivudine),FORTOVASE® (saquinavir-sg), HIVID® (zalcitabine), INVIRASE®(saquinavir-hg), KALETRA® (lopinavir-ritonavir), LEXIVA™(fosamprenavir), NORVIR® (ritonavir), RITROVIR® (zidovudine) SUSTIVA®(efavirenz), VIDEX EC® (didanosine), VIDEX® (didanosine), VIRACEPT®(nelfinavir) VIRAIVIUNE® (nevirapine), ZERIT® (stavudine), ZIAGEN®(abacavir), FUZEON® (enfuvirtide) RESCRIPTOR® (delavirdine), REYATAZ®(atazanavir), TRIZIVIR® (abacavir-lamivudine-zidovudine) VIREAD®(tenofovir disoproxil fumarate) ISENTRESS® (raltegravir), SELZENTRY®(maraviroc), and AGENERASE® (amprenavir).

Examples of viral infections include but are not limited to, infectionscaused by all RNA viruses (including negative stranded RNA viruses,positive stranded RNA viruses, double stranded RNA viruses andretroviruses) and DNA viruses. Examples of viruses include, but are notlimited to, HIV (including HIV-1 and HIV-2), parvovirus,papillomaviruses, measles, filovirus (for example, Ebola, Marburg), SARS(severe acute respiratory syndrome) virus, hantaviruses, influenzaviruses (e.g., influenza A, B and C viruses), hepatitis viruses A to G,caliciviruses, astroviruses, rotaviruses, reovirus, coronaviruses, (forexample, human respiratory coronavirus and SARS coronavirus (SARS-CoV)),picornaviruses, (for example, human rhinovirus and enterovirus), Ebolavirus, human herpesvirus (such as, HSV-1-9, including zoster,Epstein-Barr, and human cytomegalovirus), foot and mouth disease virus,human adenovirus, adeno-associated virus, respiratory syncytial virus(RSV), smallpox virus (variola), cowpox, monkey pox, vaccinia, polio,viral meningitis and hantaviruses.

For animals, viruses include, but are not limited to, the animalcounterpart to any above listed human virus, avian influenza (forexample, strains H5N1, H5N2, H7N1, H7N7 and H9N2), and animalretroviruses, such as simian immunodeficiency virus, avianimmunodeficiency virus, pseudocowpox, bovine immunodeficiency virus,feline immunodeficiency virus, equine infectious anemia virus, caprinearthritis encephalitis virus and visna virus.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Example 1 Inhibitory D-Peptides

a) Peptide Synthesis

In this example, all synthesized peptides were capped with N-terminalacetyl and C-terminal amide groups, and their masses confirmed byMALDI-TOF. Crude peptides were purified by reverse-phase HPLC (RP-HPLC)on a C18 column (Vydac) and lyophilized. D-peptide inhibitors wereoxidized at (≤0.4 mg/mL) overnight at 37° C. in 50 mM Tris, pH 8.0, 2%DMSO, and repurified using RP-HPLC.

PEG-(PIE7)₂ was made by incubating PIE7 (˜2 mM) with freshly preparedcrosslinker (Bis-dPEG₉™ NHS ester, Quanta BioDesign, #10246) at a 1:0.6(peptide:PEG) molar ratio in 50 mM NaHPO₄, pH 7.0, for 2 h at RT.PEG-(PIE2-AAA)₂ was made using a similar protocol. PEG-PIE7 was madeusing a similar protocol with NHS-m-dPEG™ (Quanta BioDesign, #10260) ata 1:2 (peptide:PEG) molar ratio.

b) Viral Infectivity Assay:

This study included the use of the HIV HXB2 strain and the HIV JRFLstrain. Viral infectivity was measured essentially as previouslydescribed (Hamburger 2005) with several modifications. 8 μg/mLDEAE-Dextran was used as a fusion enhancer in all assays.

Media was changed 22-26 h following infection. IC₅₀ values werecalculated by fitting normalized data to the following equation:y=1/(1+[inhibitor]/IC₅₀). Fits were weighted by the absolute s.e.m. fromquadruplicate points with the minimum allowable error set to 1%. Samplesrequiring DMSO for solubility (D10-p5, PEG-(PIE2-AAA)2, PEG-(PIE7)2, andPEG-PIE7) were tested at 1% final DMSO concentration and normalized toan uninhibited control containing 1% DMSO. The following reagents wereobtained through the AIDS Research and Reference Reagent Program,Division of AIDS, NIAID, TAK779 and T-20 (FUZEON®) from NIAID and Roche,respectively.

c) Results and Discussion

The peptides discussed herein are D-peptides that are pocket-specificinhibitors of entry (PIE). Examples include: PIE7-GK (GACDYPEWQWLCAAGK,SEQ ID NO: 23). This peptide is the same as PIE7, except that the Lyshas been moved to the C-terminus. The move results in slightly enhancedpotency and allows for the crosslinking of peptides via their C-termini.

PIE7-GKK (GACDYPEWQWLCAAGKK, SEQ ID NO: 24). This is a double Lysvariant of PIE7-GK, and serves as a central peptide in trimeric PIE7(the central PIE7-GKK is connected to two flanking PIE7-GK peptides).These connections are all via the C-terminus.

K-PIE7-GK (KGACDYPEWQWLCAAGK, SEQ ID NO: 25). This double Lys variant ofPIE7-GK serves as a central peptide in trimeric PIE7 (the centralK-PIE7-GK is connected to two flanking peptides—PIE7-GK and PIE7). Theseconnections link the N- to C-termini of neighboring peptides.

PIE7-GK-PEG₄: (PIE7-GK with PEG₄ attached). This peptide is a control todetermine how well PEG additions are tolerated on the C-terminus of theD-peptides. From this peptide, it was learned that such additions arewell tolerated.

The following group of new peptides arose from optimization of theflanking sequences:

(SEQ ID NO: 26) HPCDYPEWQWLCELGK (SEQ ID NO: 27) HPCDYPEWQWLCKLGK(SEQ ID NO: 28) HPCDYPEWQWLCRLGK (SEQ ID NO: 29) HACDYPEWQWLCELGK

The multimers disclosed herein were found to have avidity. Moreparticularly, the dimeric inhibitors PEG-(PIE2-AAA)₂ and PEG-(PIE7)₂have IC_(50s) of 21 nM and 1.9 nM (Table 2), respectively. These valuesrepresent a ˜70- and ˜325-fold improvement over the correspondingmonomers. These data also indicate that modest improvements in thepotency of monomeric inhibitors are magnified by avidity in the dimer,as also observed in the phage display. The potency of PEG-(PIE7)₂ iscomparable to FUZEON® (Table 2). The improved potency of the dimerscannot be attributed to an interaction of the PEG with virus, cells, orthe D-peptide, but is a genuine avidity effect caused by two D-peptidesbinding to the N-trimer.

TABLE 2 Inhibitory Potency of D-peptides against HXB2 and JRFLPseudovirion Entry IC₅₀ (nM)^(†) IC₅₀ (nM) Sample (HXB2) (JRFL) D10-p59.5 >25,000 2K-PIE1 2.2 >100,000 2K-PIE2 2.6 ND PIE2-AAA 1.4 ND PIE2 1.366,000 PIE8 1.7 ~110,000 PIE7 620 24000 PIE7-GK 390 15800 PIE7-GKK 38019100 K-PIE7-GK ND 14800 PIE7-GK-PEG₄ 350 13100 PIE12 37 578 PIE13 411470 PIE14 33 1050 PIE15 67 1450 N₉N(PIE7)₂ 1.9 2300 N₉C(PIE7)₂ 0.33 310C₉C(PIE7-GK)₂ 0.35 220 N₅N(PIE7)₂ 1.1 1410 N₅C(PIE7)₂ 0.58 300C₅C(PIE7-GK)₂ 0.36 200 N₀N(PIE7)₂ 0.80 1090 N₀C(PIE7)₂ 0.53 400C₀C(PIE12)₂ 0.29 14 PEG5-(PIE13)₂ ND 15 N₉N(PIE7)₃ 0.25 220C₅C(PIE7-GK)₃ 0.13 6.7 C₅C(PIE12)₃ ND 2.8 C₀C(PIE7-GK)₃ 0.12 16.1PIE7-GK long claw 0.12 20.6 PIE7-GK short claw 0.23 86.6 C37 1.4 13.0FUZEON ® 3.7 5.0 ^(†)IC₅₀ s.e.m. is <20% and K_(D) s.e.m. is <5% forduplicate assays for all values ND = Not determined

The use of the peptides disclosed herein have significant effects acrossHIV clades utilizing the same PIE construct. For example, PIE12-trimershowed IC₅₀ of less than 10 nM against replication competent HIV strainsthat cover envelope subtypes A, B, C, D, E, F, and G while maintainingno toxicity for dosage up to 10 μM (Table 3). By comparison, the IC₅₀ ofFUZEON® was less than the 10 nM level in only three strains (clades Band E only) and in those instances was between 6 and 100 times lesspotent than the PIE12-trimer. In fact, FUZEON® had >100-fold lessinhibition for HIV subtype A viruses, from 6- to 250-fold lessinhibition for subtype B viruses, ˜60-fold less inhibition for subtype Cviruses, between 40- and 100-fold less inhibition for subtype D viruses,25- to 150-fold less inhibition for subtype F strains, and 20 to100-fold less inhibition for subtype G strains.

TABLE 3 Inhibitory breadth against replication competent virus and PBMCtarget cells. HIV-1 Envelope IC50 (nM) Isolate Subtype PIE12 trimerFuzeon IIIB B 0.78 28.3 Ba-L B 0.27 19.7 JR-CSF B 0.08 7.03 JR-FL B 0.261.65 92UG029 A 0.69 191 92UG037 A 0.21 41.45 93IN101 C 0.38 22.4 92BR025C 5.19 305 92UG001 D 4.45 182 92UG046 D 1.2 130 CMU02 AE 0.35 43.893TH073 E 0.84 198 CMU06 E 0.38 5.65 93BR019 BF 4.72 >1,000 93BR020 F0.37 58.5 93BR029 F 0.79 18.6 G3 G 1.21 23.3 RU570 G 0.36 37 No toxicityobserved up to 10 μM

Example 2 Cholesterol Cargo

a) Synthesis of D-peptides with Cholesterol Cargo

D-peptide monomers with cholesterol potency-enhancing cargo were madeusing heterobifunctional PEG NHS ester/maleimide crosslinkers toconjugate thiocholesterol (cholesterol with a thiol replacing itshydroxyl group) to the terminal Lys of PIE12-GK (where PEG ispolyethylene glycol and NHS is N-Hydroxysuccinimide; Quanta BioDesign,catalog #10994). For synthesis of D-peptide trimers withpotency-enhancing cholesterol cargo, PIE12-GK (11 mM) was reacted withFmoc-N-amido-dPEG₄-NHS ester at a 1.05:1 (peptide:PEG) molar ratio indimethylacetamide buffered by triethylamine (TEA, 200 mM) for 40 minutesat room temperature (RT). The product was purified by C18 reverse phase(RP) HPLC and lyophilized. The Fmoc protecting group was then removed byresuspending the product in 20% piperidine (in dimethylacetamide), andthe deprotected product was purified by C18 RP-HPLC. This material(PIE12-GK-PEG4-NH2, 10 mM) was reacted with Mal-dPEG₁₂-Tris(NHS)₃(Quanta BioDesign, catalog #10676) at a 3.2:1 (peptide:scaffold) molarratio in dimethylacetamide buffered by 200 mM triethylamine at RT for 40min. Reaction progress was monitored by RP-HPLC. To this reaction,thiocholesterol (25 mM stock solution in 90% Dimethylacetamide, 10%chloroform, 200 mM TEA) was added to a final concentration of 4 mM. Thefinal product was purified by RP-HPLC and lyophilized, and its identityconfirmed by mass spectroscopy (mass 8061.81 Da).

Lengths of the three NHS ester arms (used for attaching PIEs) can bemodified by using Fmoc-N-amido-dPEGx-NHS ester, where x can range from 1to 36 (as discrete PEG lengths) or longer (as polydisperse PEGs with aspecified average length). PEGs can be synthesized as continuousmolecules or can be stitched together using smaller PEGs. For example,reacting the maleimide group on the PEG₁₂ arm with a cysteine residueconverts the maleimide to a primary amine, which can be used with NHSester PEG-maleimide to extend the length of the PEG arm and restore amaleimide group to its terminus. This was the strategy used to make PEGsdesignated with an asterisk in Table 4.

b) Results

Table 4 shows the results of conjugating the cholesterol cargo to PIE12monomer or PIE12-trimer on the inhibition of membrane entry by two HIVstrains, HXB2 and JRFL. For the PIE12 monomer, addition of chol-PEG₂resulted in a significantly weaker inhibitor, while a PEG₁₂ linkerprovided a nearly 3-fold improvement in potency. Unexpectedly, a PEG₂₄linker provided a ˜100-fold boost in potency for the PIE12 monomer.Application of PEG₂₄ to PIE12-trimer resulted in an even greaterimprovement in potency (˜250-fold). In Table 4, the asterisks refer toPEG chains that were assembled by connecting two smaller PEG chains(e.g., PEG₂₄*=two PEG12 chains connected in series).

TABLE 4 Inhibitory Potency of D-peptides against HXB2 and JRFLPseudovirion Entry with cholesterol potency-enhancing cargo. IC50 IC50(nM) IC50 (nM) (nM) IC50 (nM) E560K/ Q577R Sample (HXB2) (JRFL)V570IHXB2 HXB2 PIE12 37 580 240 10200 Chol-PEG₂-PIE12 58 (monomer)Chol-PEG₁₂- 11 200 PIE12(monomer) Chol-PEG₂₄-PIE12 0.40 (monomer)C5C-PIE12-trimer 0.33 2.8 0.84 2960 Chol-PEG₁₂-PIE12- 0.044 trimerChol-PEG₁₆*-PIE12- 0.020 trimer Chol-PEG₂₄*-PIE12- 0.011 trimerChol-PEG₃₆*-PIE12- 0.0054 0.011 0.0045 2.7 trimer Chol-PEG₂₄-PIE12-0.0142 trimer

As shown in FIG. 1 and FIG. 2, the gp41 pocket region is approximately˜57 Å to ˜60 Å away from the cell surface. Therefore, a relatively longlinker may be used to bridge this distance between the membrane and aPIE (e.g., PIE12 in FIG. 2). Prior to the testing conducted herein, itwas thought that such a long linker arm could reduce the localconcentrating effect of cholesterol. Surprisingly, this is not the case.As seen in Table 4, cholesterol connected to monomeric or trimeric PIE12via long PEG arms (e.g., 24 or 36 units, predicted to be long enough tobridge 57 Å) produces an inhibitor with >100-fold improved potency.

Table 4 also shows two resistance mutations discovered againstPIE7-dimer (E560K/V570I) and PIE12-trimer (Q577R). The resistancemutations have a similar relative effect on cholesterol linkedPIE12-trimers, but the much improved potency of the cholesterol-linkedpeptides allows these peptides to maintain good inhibitory activity evenagainst these resistant strains.

Example 3 Modular Homotrimeric Scaffold

A homotrimeric scaffold was designed containing three NHS ester arms(trimeric trimethoylester triNHS) for conjugation to D-peptides in asingle-pot reaction. PEG linkers of various lengths can be appended tothe D-peptide, allowing for the simple production of D-peptide-trimerswith varying PEG lengths.

a) Peptide Synthesis

Peptides were synthesized using a PTI PS3 peptide synthesizer or by RSSynthesis to generate either PIE12-GK or AHP-PIE12-GK (lacks twoN-terminal residues, D-His and D-Pro) (Welch, 2007 and 2010).PIE12-dPEG_(4/5)-NH₂ (the precursor to PIE12-trimer synthesis) wassynthesized as follows: PIE12-GK (10 mM in dimethylacetamide, DMAC) wasreacted with 250 mM Fmoc-N-amido-dPEG_(4/5)-NHS ester (Quanta BioDesign10994 and 10053) in dry DMAC (Acros Organics, septa sealed withmolecular sieves) at a 1:1 molar ratio buffered by triethylamine (200mM, pH 7.5) for 60 minutes at RT. This reaction was quenched by additionof acetic acid to 5% and purified by reverse-phase HPLC(water/acetonitrile gradient in 0.1% TFA) on a Waters BEH X-Bridge 10μm, 300 Å C₁₈ column (RP-HPLC). Purified product was lyophilized, thenresuspended in 20% piperidine in DMAC for 20 minutes to remove Fmoc andproduce PIE12-PEG_(4/5)-NH₂, which was then purified by RP-HPLC.

b) Trimer Synthesis

PIE12-PEG_(4/5)-NH₂ (10 mM) was reacted with 250 mMtrimethylolethane-triNHS ester (FIG. 3a , Quanta BioDesign 10674) inDMAC at a 3.3:1 (peptide/scaffold) ratio in DMAC buffered bytriethylamine (200 mM, pH 7.5) for 60 minutes at RT. Product waspurified by RP-HPLC. All masses were confirmed by ESI-MS (AB SciexAPI-3000).

Cholesterol-PIE12-trimer and alkyl-PIE12-trimer were synthesized asfollows: PIE12-PEG₄-NH₂ (10 mM) was reacted with Maleimide-PEG₁₂-triNHSester (Quanta BioDesign 10676, 250 mM in DMAC) or Maleimide-PEG₂₄-triNHSester (FIG. 3b , Quanta BioDesign 10680, 250 mM in DMAC) at a 3.3:1(peptide/scaffold) ratio in DMAC buffered by triethylamine (200 mM, pH7.5) for 45 minutes at RT, each using a carbon-centered multimerscaffold. Thiocholesterol (Sigma Aldrich, 136115, 250 mM in chloroform),1-octanethiol (Sigma-Aldrich 471836), 1-Hexadecanethiol (Sigma-Aldrich52270) or 1-octadecanethiol (Sigma Aldrich 01858) were then added to afinal concentration of 4.5 mM and reacted for an additional 60 minutes.For PEG₁₆, PIE12-PEG₄-NH₂ was first reacted with Mal-PEG₁₂-triNHS ester,followed by reaction with D-Cysteine (5 mM) to yield(PIE12-PEG₄)₃-PEG₁₂-Cys. This product was then purified by RP-HPLCbefore sequential reaction with Maleimide-PEG₄-NHS and thiocholesterolunder conditions identical to those used to generatechol-PEG₂₄-PIE12-timer. PEG₃₆, PEG₅₇, and PEG₁₃₂-trimer were producedthrough conjugation of PIE12-PEG₄-NH₂ to Maleimide-PEG₂₄-triNHS ester,followed by addition of D-Cysteine. This intermediate was thenconjugated to Mal-PEG₁₂-NHS ester (Quanta Biodesign, 10284),Mal-PEG_(2K)-NHS ester (Creative PEGWorks, PHB-950, ˜45 PEG units), orMal-PEG_(5K)-NHS ester (Creative PEGWorks, PHB-952, ˜120 PEG units) toyield Chol-PEG₃₆-PIE12-Trimer, Chol-PEG₅₇-PIE12-trimer, andChol-PEG₁₃₂-PIE12-trimer, respectively. The reaction was quenched byaddition of acetic acid to 5% before purification by RP-HPLC.

c) Viral Infectivity Assays

Pseudovirion infectivity assays were carried out using HIV HXB2 strainand HIV JRFL strain luciferase reporter pseudovirions (NL4-3 strain) andHOS-CD4-CXCR4 (for HXB2) or HOS-CD4-CCR5 (for JRFL) target cells (Welch,2007 and 2010). Inhibitors curves were generated using six concentrationpoints measured in quadruplicate, and luciferase counts were normalizedto an uninhibited control. Inhibition curves were fit using a standardIC₅₀ equation (1−c/(IC₅₀+c)) weighting each concentration point by itsstandard error in KaleidaGraph™ (Synergy Software). Reported IC₅₀ valuesare the average of at least 2 independent assays.

d) Results

Results show the successful synthesis of D-peptide-trimer using ahomotrimer scaffold while also optimizing the PEG linkages betweenD-peptide monomers. Previously, PIE12-trimer had been synthesized byattaching bis-NHS ester PEG₅ spacers to PIE12-GK. After purification,two of these PEGylated monomers were reacted with a central PIE12-GKKmonomer (two primary amines) to produce PIE12-trimer (Welch, 2010). Thismethod is cumbersome for large-scale production because it requires thesynthesis of two distinct D-peptides and a series of HPLC purificationsto assemble the trimer, resulting in low yields. In addition, the PIE12crystal structure suggested that shorter PEG linkers might adequatelybridge the neighboring pockets and improve avidity. To overcome theshort comings of this method, a homotrimeric scaffold was designedcontaining three NHS ester arms for conjugation to D-peptide monomers ina single pot reaction, such as conjugation with PIE12-GK (FIG. 3a ). PEGlinkers of various lengths can be appended to the PIE12-GK peptide,allowing for the simple production of PIE12-trimers with varying PEGlengths.

PIE12-trimer's estimated sub-fM affinity for the N-trimer makes directcomparative K_(D) measurements (e.g., by surface plasmon resonance)challenging. Although antiviral potency can be used as a surrogate foraffinity, PIE12-trimer's potency plateau can mask even large changes inaffinity. To overcome this problem, a PIE12 variant was designed withweakened affinity to allow comparative evaluation of different trimergeometries by measuring inhibitor potency. PIE12's two N-terminalresidues make important contacts with the N-trimer and it was reasonedthat deletion of these residues (D-His and D-Pro) would significantlyreduce binding affinity without disrupting the overall orientation ofPIE12 binding to the gp41 pocket or the local structure at theC-terminal PEG linkage site. AHP-PIE12 is 84-fold less potent than PIE12(Table 5). In the context of the homotrimeric scaffold, AHP-PIE12connected via PEG₅ linkers has an IC₅₀ of 380 nM against HXB2 (astandard lab-adapted strain) and is therefore well outside of thepotency plateau (˜500 pM for HXB2). Using AHP-PIE12-trimer, changescould be detected in potency due to linker changes that subtly alteraffinity.

TABLE 5 D-peptide Inhibition data IC₅₀ (nM) Inhibitor HXB2 JRFL PIE12*37 ± 2.3   580 ± 21.4 ΔHP-PIE12 3100 ± 783  nd Chol-PEG₂-PIE12-monomer69 ± 11  nd Chol-PEG₁₂-PIE12-monomer 12 ± 3.6  ndChol-PEG₂₄-PIE12-monomer 0.64 ± 0.25  nd C34 1.4 ± 0.3  13.4 ± 0.1 C34-PEG₂-Chol 0.044 ± 0.0004 0.05 ± 0.01 C34-PEG₁₁-Chol 0.021 ± 0.00140.024 ± 0.005 C34-PEG₈₀-Chol 0.022 ± 0.0004  0.1 ± 0.045PEG₄-ΔHP-PIE12-trimer 300 ± 7.2   nd PEG₅-ΔHP-PIE12-trimer 380 ± 13  ndPEG₄-PIE12-trimer 0.72 ± 0.04   2.1 ± 0.28 Chol-PEG₁₂-PIE12-trimer 0.052± 0.02   0.06 ± 0.004 Chol-PEG₁₆-PIE12-trimer 0.02 ± 0.002  0.017 ±0.0002 Chol-PEG₂₄-PIE12-trimer 0.013 ± 0.0013 0.019 ± 0.003Chol-PEG₃₆-PIE12-trimer 0.011 ± 0.0015 0.015 ± 0.005Chol-PEG₅₇-PIE12-trimer 0.007 ± 0.0013 0.013 ± 0.003Chol-PEG₁₃₂-PIE12-trimer 0.012 ± 0.0015 0.025 ± 0.002C8-PEG₂₄-PIE12-trimer 0.42 ± 0.01  nd C16-PEG₂₄-PIE12-trimer 0.09 ±0.014  0.11 ± 0.012 C18-PEG₂₄-PIE12-trimer 0.054 ± 0.018  0.087 ± 0.012Antiviral potency against HXB2 and JRFL HIV-1 strains (*from 20).

Example 4 Modular Heterotetrameric Scaffold and PEG Linker Optimization

To enable the conjugation of potency-enhancing cargo groups toPIE12-trimer, a heterotetrameric scaffold was designed. Theheterotetrameric scaffold comprised three shorter PEG arms with NHSester groups, for addition of PIE12-PEG₄-NH₂, a carbon center, and afourth PEG arm of variable length functionalized with maleimide, anorthogonal reactive group, for the addition of potency-enhancing cargoes(i.e. pharmacokinetic-enhancing and/or membrane-localizing cargoes (FIG.3b ).

One of the potency-enhancing cargoes studied for the heterotetramericscaffold was cholesterol. As disclosed herein, a consideration for usinga cholesterol cargo with PIE12 D-peptide is that while the N-terminus ofthe C-peptide lies immediately adjacent to the membrane, PIE12 targets apocket that is ˜60 Å from the cell membrane (see, e.g. FIG. 1). FlexiblePEG linkers of varying lengths were used to span this distance. PEG₁₂ issufficiently long if stretched taut, but PEG typically assumes anaverage length approximately half of its fully stretched distance.

To study the potency effects of cholesterol (chol) conjugation to aD-peptide, and to optimize the length of the PEG linker between chol anda D-peptide, monomeric PIE12 was used, which is not in a potency plateauand therefore should be a sensitive reporter for optimal PEG linkerlength. Chol-PEG_(x)-PIE12 conjugates were generated usingheterobifunctional PEG₂, PEG₁₂, and PEG₂₄ NHS ester/maleimidecrosslinkers to conjugate thiocholesterol (cholesterol with a thiolreplacing its hydroxyl group) to PIE12's C-terminal Lys (its onlyprimary amine). It was observed that the chol-PEG₂-PIE12 conjugate wastoo short to bridge the membrane-to-pocket distance and caused atwo-fold loss of potency (HXB2 strain) compared to unconjugated PIE12(Table 5). In contrast, chol-PEG₁₂-PIE12 shows 3-fold improved potency,while chol-PEG₂₄-PIE12 provided an even greater 58-fold increase inpotency compared to PIE12 alone (Table 5).

For comparison, C-peptide (C34) cholesterol conjugates of varyinglengths were synthesized (Table 5). Results showed a ˜40-fold improvedpotency using a short PEG₂ linker, but surprisingly, a longer linker(PEG₁₁) provides an additional 2-fold improvement in potency, and a muchlonger linker (PEG₈₀) maintains the same potency (HXB2 strain). Asimilar pattern is seen with the JRFL strain, but with significantattenuation at very long PEG linker lengths (4-fold worse than theoptimal PEG length).

Based on these potency gains, PIE12-trimer was conjugated to cholesterolusing the heterotetrameric scaffold as disclosed herein. Using theoptimal PEG₄ linker determined earlier for the three NHS ester (PIE12)arms, chol-PIE12-trimers were synthesized with a variety of fourth arm(maleimide) lengths to confirm the relationship between PEG length andpotency observed with the monomer. This sensitivity was expected becausemembrane localization affects the association rate rather than changingaffinity (masked by the resistance capacitor). The length of the fourtharm was varied from 12 to 132 PEG units, covering a distance range of˜60 to ˜480 Å (fully-extended).

Cholesterol conjugation dramatically improved PEG₄-PIE12-trimer potencyagainst both HXB2 and JRFL entry (up to 160-fold, Table 5). Comparisonof varying 4^(th) arm lengths in chol-PIE12-trimer shows that inhibitorpotency varies modestly in an optimal range between PEG₂₄ and PEG₅₇. Asshown in FIG. 4a , a shorter PEG₁₂ linker is less potent than PEG₂₄ andPEG₃₆ linkers. Only a slight decrease in potency is observed with thelongest (PEG₁₃₂) linker (not shown). The chol-PEG₂₄-PIE12-trimer waschosen for further study due to its ease of synthesis and theavailability of monodisperse PEG₂₄. Furthermore, a monodisperse PEG₂₄scaffold may ease preclinical studies of chol-PIE12-trimer purity,metabolism, pharmacokinetics, and stability. Importantly, cholesterolconjugates retain high (mM) aqueous solubility.

Another strategy for potency enhancement and localizing D-peptideinhibitors to membranes is the use of the heterotetrameric scaffold withfatty acid cargo at the fourth arm. Using the same heterotetramerscaffold synthesis strategy described herein, PIE12-trimers weresynthesized and conjugated at the fourth arm to fatty acids cargoes withaliphatic chains of either 8, 16, or 18 carbons (C8/C16/C18 fattyacid-PEG₂₄-PIE12-trimer). While C8 fatty acid conjugation has littleeffect on PIE12-trimer potency, C16 fatty acid and C18 fatty acid bothprovide gains in potency, though to a lesser degree than seen withcholesterol (FIG. 4b and Table 5). C18 fatty acid-PEG₂₄-PIE12-trimer wasslightly more potent than C16 fatty acid-PEG₂₄-PIE12-trimer (FIG. 4b ).

These results demonstrate the successful application of modular multimerscaffold-based design to peptide drug optimization (both peptidegeometry and localization to the site of action via conjugated membranelocalizing cargoes). This approach allows for alterations in thescaffold to accommodate a variety of cargoes and chemistries (e.g.,“click” chemistry), as well as rapid optimization of PEG linker lengths.For viruses that undergo membrane fusion within the endosome, such asEbola, this strategy could be employed to attach an endosome-targetingmoiety to localize an inhibitor to the site of virus entry and increaseinhibitor potency. Additionally, the multimer scaffold allows forD-peptide conjugation to a variety of cargoes to modulate potency,pharmacokinetic properties (e.g., large branched PEGs, albumin, oralbumin-binding peptides), and membrane localization.

Example 5 Effect of Membrane Localization on the Resistance Capacitor

Drug resistance is a constant threat to the effectiveness of HIVinhibitors. PIE12-trimer and its variants are an attractive drugcandidates in part because of a strong resistance capacitor, whichprovides a high genetic barrier to resistance (Welch, 2010). Theresistance capacitor depends on the diffusion-limited association ratefor PIE12-trimer binding to gp41. The cholesterol and C16/18 alkanechain conjugation strategies described herein break through this kineticbarrier via inhibitor localization to viral entry sites (i.e.,increasing effective inhibitor concentration and overcoming thediffusion rate limitation). In theory, this improvement in potency couldcome at the cost of weakening the resistance capacitor. To test for thispossibility, the potency of chol- and C16/C18 alkane chain-conjugatedPIE12-trimer was measured against previously identified resistancemutations (Welch, 2010).

Previous selection for resistance to PIE7-dimer (an earlier-generationD-peptide inhibitor) generated E560K/V570I, which minimally affects thepotency of PIE12-trimer, but dramatically reduces PIE7-dimer potency.Selection of resistance to PIE12-trimer required more than a year ofviral passaging, but ultimately resulted in the Q577R mutation, whichdecreases PIE12-trimer potency by >1000-fold (Welch, 2010). The effectof these resistance mutations on chol- and C16/18 alkanechain-PIE12-trimer potency is shown in Table 6. The relative effects ofboth resistance mutations are similar for PIE12-trimer and thecholesterol/alkane-conjugated PIE12-trimers. However, because of thegreatly improved potency of the conjugated PIE12-trimers, theseinhibitors maintain nM potency even against the severe Q577R resistancemutation. The impact of the less severe E560K/V570I resistance mutationis absorbed by all of the conjugated PIE12-trimers, as well as plainPIE12-trimer. These data suggest that the improvement in potency throughC16/C18 alkane chain and cholesterol conjugation retains enough excessbinding energy to maintain an effective resistance capacitor.

TABLE 6 Antiviral potency against resistant strains. IC₅₀ (nM) InhibitorWT HXB2 E560K/V570I Q577R PEG₄-PIE12-trimer 0.72 0.89 >3 μMChol-PEG₂₄-PIE12-trimer 0.013 0.01 10.1 C8-PEG₂₄-PIE12-trimer 0.42 0.86452 C16-PEG₂₄-PIE12-trimer 0.09 0.045 39 C18-PEG₂₄-PIE12-trimer 0.0540.035 32.5 Antiviral potency against identified resistant strains (HXB2background). The IC₅₀ standard error of the mean values are <35% for allsamples.

Example 6 Pharmacokinetic Properties of D-peptide Inhibitors

Although PIE12-trimers without a potency-enhancing cargo has desiredantiviral properties, its relatively small size (˜8 kD) may lead to ashort serum half-life due to renal filtration. In addition to theirpotency-boosting effects, it was hypothesized that both cholesterol andalkyl conjugation will also lead to improvements in the pharmacokinetic(PK) properties, or pharmacokinetic enhancement, of these D-peptideinhibitors via interaction with cell membranes and albumin that slowrenal clearance. Albumin serves as a carrier for both cholesterol andfatty acids, reducing the rate of renal filtration. Adherence tomembrane surfaces may also slow the absorption of D-peptide inhibitorfrom the subcutaneous space, enabling prolonged dosing via aslow-release depot effect. This type of depot would be especiallyattractive for non-degradable D-peptides.

To evaluate the PK properties of the cholesterol conjugated D-peptides,the chol-PIE12-trimer and the PIE12-trimer were administeredsubcutaneously to rats (1 mg/kg). After subcutaneous administration, itwas observed that the chol-PIE12-trimer with cholesterol conjugated witha PEG₂₄ linker shows an enhanced plasma half-life over the PIE12-trimer.More specifically, the chol-PIE12-trimer has a longer half-life of 3.7hours compared to 2.7 hours for the PIE12-trimer. Furthermore, thechol-PIE12-trimer shows a reduced clearance of 24 mL/hour/kg compared to572 mL/hr/kg for PIE12-trimer. As shown in FIG. 5, the chol-PIE12-trimershows an improved concentration-time profile compared to thePIE12-trimer following subcutaneous administration in rats.

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What is claimed is:
 1. A composition comprising at least threeD-peptides and at least one potency-enhancing cargo molecule linked to atetrameric scaffold, wherein: each D-peptide comprises the sequence ofSEQ ID NO:37 (CDYPEWQWLC); and the potency-enhancing cargo molecule is amembrane localizing potency enhancing cargo molecule selected from thegroup consisting of a cholesterol or an analog thereof, alkane chain,and a fatty acid, and the membrane localizing potency-enhancing cargomolecule is linked to the tetrameric scaffold via a polyethylene glycol(PEG) linker comprising 12 to 132 PEG units.
 2. The composition of claim1, wherein the potency-enhancing cargo molecule is cholesterol orthiocholesterol.
 3. The composition of claim 1, wherein thepotency-enhancing cargo molecule is an alkane chain.
 4. The compositionof claim 3, wherein the potency-enhancing cargo molecule is a C8 alkane,a C16 alkane, or a C18 alkane.
 5. The composition of claim 1, whereinthe potency-enhancing cargo molecule is a fatty acid.
 6. The compositionof claim 5, wherein the potency-enhancing cargo molecule is a C8 fattyacid, a C16 fatty acid, or a C18 fatty acid.
 7. The composition of claim1, wherein each D-peptide is identical.
 8. The composition of claim 1,wherein at least two D-peptides are different.
 9. The composition ofclaim 1, wherein at least one D-peptide comprises the amino acidsequence of any one of SEQ ID NOS:6 and 23-29.
 10. The composition ofclaim 1, wherein each D-peptide comprises an amino acid sequence of SEQID NO:26.
 11. The composition of claim 1, wherein the tetramericscaffold is a heterotetrameric scaffold comprising three NHS estergroups and a fourth orthogonal group, wherein the at least threeD-peptides are linked to the heterotetrameric scaffold via the three NHSester groups and the potency-enhancing cargo molecule is linked to theheterotetrameric scaffold via the fourth orthogonal group, wherein thefourth orthogonal group comprises the PEG linker comprising 12 to 132PEG units.
 12. The composition of claim 1, wherein the PEG linker isPEG12, PEG16, PEG24, PEG25, PEG26, PEG27, PEG28, PEG29, PEG30, PEG31,PEG32, PEG33, PEG34, PEG35, PEG36, PEG57 or PEG132.
 13. The compositionof claim 1, wherein the tetrameric scaffold comprises a tris, di-lysine,benzene ring, phosphate, or peptide core.
 14. The composition of claim11, wherein the potency enhancing cargo molecule is joined to the PEGlinker via a reactive group.
 15. The composition of claim 14, whereinthe reactive group is a maleimide reactive group.
 16. The composition ofclaim 11, wherein the tetrameric scaffold comprises a structure asfollows:


17. The composition of claim 1, wherein the composition inhibits HIVentry into a cell.
 18. A pharmaceutical composition comprising thecomposition of claim 1 and a pharmaceutically acceptable carrier.